Method of reducing tumor relapse rate in immunotherapy by administration of lenzilumab

ABSTRACT

Methods for reducing relapse rate or preventing occurrence of tumor relapse in a subject treated with immunotherapy, in an absence of an incidence of immunotherapy-related toxicity or in a presence of immunotherapy-related toxicity. Methods for reducing a level of a cytokine or chemokine other than GM-CSF in a subject having an incidence of immunotherapy-related toxicity, the methods comprising administering a recombinant GM-CSF antagonist to the subject. Methods for treating or preventing immunotherapy-related toxicity in a subject, the methods comprising administering to the subject chimeric antigen receptor-expressing T-cells (CAR-T cells), the CAR-T cells having a GM-CSF gene knockout (GM-CSF k/o  CAR-T cells), and a recombinant hGM-CSF antagonist.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of U.S.application Ser. No. 16/204,220, filed on Nov. 29, 2018, which is acontinuation-in-part application of U.S. application Ser. No.16/149,346, filed on Oct. 2, 2018, which claims priority to U.S.Provisional Application Nos. 62/567,187, filed Oct. 2, 2017, and62/729,043, filed Sep. 10, 2018, which are hereby incorporated byreference.

FIELD OF THE DISCLOSURE

The invention relates to methods for reducing relapse rate or preventingoccurrence of tumor relapse in a subject treated with immunotherapy inan absence of an incidence of immunotherapy-related toxicity. Theinvention also relates to methods for reducing relapse rate orpreventing occurrence of tumor relapse in a subject treated withimmunotherapy in a presence of an incidence of immunotherapy-relatedtoxicity. The invention further relates to methods for reducing a levelof a cytokine or chemokine other than GM-CSF in a subject having anincidence of immunotherapy-related toxicity, the methods comprisingadministering a recombinant GM-CSF antagonist to the subject. Theinvention also relates to methods for treating or preventingimmunotherapy-related toxicity in a subject, the method comprisingadministering to the subject chimeric antigen receptor-expressingT-cells (CAR-T cells), the CAR-T cells having a GM-CSF gene knockout(GM-CSF^(k/o) CAR-T cells), and a recombinant hGM-CSF antagonist. Thedisclosure herein also provides methods of inhibiting or reducing theincidence and/or the severity of immunotherapy-related toxicity in asubject, the method comprising administering a recombinant GM-CSFantagonist to the subject.

BACKGROUND

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokinesecreted by various cell types including macrophages, T cells, mastcells, natural killer cells, endothelial cells and fibroblasts. GM-CSFstimulates the differentiation of granulocytes and of monocytes.Monocytes, in turn, migrate into tissue and mature into macrophages anddendritic cells. Thus, secretion of GM-CSF leads to a rapid increase inmacrophage numbers. GM-CSF is also involved in the inflammatory responsein the Central Nervous System (CNS) causing influx of blood-derivedmonocytes and macrophages, and the activation of astrocytes andmicroglia. Immuno-related toxicities comprise potentiallylife-threatening immune responses that occur as a result of the highlevels of immune activation occurring from different immunotherapies.Immuno-related toxicity is currently a major complication for theapplication of immunotherapies in cancer patients. Chimeric antigenreceptor T (CAR-T) cell therapy has emerged as a novel and potentiallyrevolutionary therapy to treat cancer. Based on unprecedented responsesin B cell malignancies, two CD19 targeted CAR-T (CART19) cell productswere approved by the FDA in 2017. However, the wider application ofCAR-T cell therapy is limited by the emergence of unique and potentiallyfatal toxicities. These include the development of cytokine releasesyndrome (CRS) and neurotoxicity (NT). Up to 50% of patients treatedwith CART19 cells develop grade 3 or higher CRS or NT and several deathshave been reported. These toxicities are associated with prolongedhospitalization, intensive care unit (ICU) stays, and the long-termeffects of NT are unknown. Thus, controlling these CART19 cell relatedtoxicities is imperative to lessen morbidity, mortality, duration ofhospitalization, ICU admissions, supportive care required and thesignificant indirect costs associated with CAR-T cell therapy. It isclear that there remains a critical need for methods of preventing andtreating immuno-related toxicity. An ideal method will minimize the riskof these life-threatening complications without affecting the efficacyof the immunotherapy and could potentially even improve the efficacy byallowing, for example, safe increased dosing of immunotherapeuticcompounds and/or an expansion of T cells.

BRIEF SUMMARY OF THE INVENTION

In one aspect, this invention provides methods for reducing relapse rateor preventing or delaying occurrence of tumor relapse in a subjecttreated with immunotherapy in an absence of an incidence ofimmunotherapy-related toxicity, the method comprising administering tothe subject a recombinant hGM-CSF antagonist. In a related aspect, thisinvention provides methods for reducing relapse rate or preventingoccurrence of tumor relapse in a subject treated with immunotherapy in apresence of an incidence of immunotherapy-related toxicity, the methodcomprising administering to the subject a recombinant hGM-CSFantagonist.

In another aspect, this invention provides a method reducing a level ofa cytokine or chemokine other than GM-CSF in a subject having anincidence of immunotherapy-related toxicity, the method comprisingadministering to the subject a recombinant hGM-CSF antagonist, whereinthe level of the cytokine or chemokine is reduced compared to the levelthereof in a subject during the incidence of immunotherapy-relatedtoxicity.

In a further aspect, this invention provides a method for treating orpreventing immunotherapy-related toxicity in a subject, the methodcomprising administering to the subject chimeric antigenreceptor-expressing T-cells (CAR-T cells), the CAR-T cells having theirGM-CSF genes ‘knocked-out’ (GM-CSF^(k/o) CAR-T cells), and a recombinanthGM-CSF antagonist.

In one aspect, disclosed herein is a method of inhibiting or reducingthe incidence or the severity of immunotherapy-related toxicity in asubject, the method comprising a step of administering a recombinanthGM-CSF antagonist to the subject.

In a related aspect, said immunotherapy comprises adoptive celltransfer, administration of monoclonal antibodies, administration ofcytokines or chemokines, administration of a cancer vaccine, T cellengaging therapies, or any combination thereof.

In another aspect, adoptive cell transfer comprises administeringchimeric antigen receptor-expressing T-cells (CAR T-cells), T-cellreceptor (TCR) modified T-cells, tumor-infiltrating lymphocytes (TIL),chimeric antigen receptor (CAR)-modified natural killer cells, ordendritic cells, or any combination thereof. In a related aspect, themonoclonal antibody is selected from a group comprising: anti-CD3,anti-CD52, anti-PD1, anti-PD-L1, anti-CTLA4, anti-CD20, anti-BCMAantibodies, bi-specific antibodies, or bispecific T-cell engager (BiTE)antibodies, or any combination thereof. In a related aspect, thecytokines are selected from a group comprising: IFNα, IFNβ, IFNγ, IFNλ,IL-1, IL-2, IL-6, IL-7, IL-15, IL-21, IL-11, IL-12, IL-18, GM-CSF, TNFα,or any combination thereof.

In another aspect, inhibiting or reducing the incidence or the severityof immunotherapy-related toxicity comprises reducing the concentrationof at least one inflammation-associated factor in the serum, tissuefluid, or in the CSF of the subject. In a related aspect, theinflammation-associated factor is selected from a group comprising:C-reactive protein, GM-CSF, IL-1, IL-2, sIL2Rα, IL-5, IL-6, IL-8, IL-10,IP10, IL-15, MCP-1 (AKA CCL2), MIG, MIP1β, IFNγ, CX3CR1, or TNFα, or anycombination thereof. In another aspect, the administration ofrecombinant GM-CSF antagonist does not reduce the efficacy of saidimmunotherapy. In another aspect, the administration of recombinantGM-CSF antagonist increases the efficacy of said immunotherapy. Inanother aspect, administration of recombinant GM-CSF antagonist occursprior to, concurrent with, or following immunotherapy. In a relatedaspect, the recombinant GM-CSF antagonist is co-administered withcorticosteroids, anti-IL-6 antibodies, tocilizumab, anti-IL-1antibodies, cyclosporine, antiepileptic s, benzodiazepines,acetazolamide, hyperventilation therapy, or hyperosmolar therapy, or anycombination thereof.

In another aspect, the immunotherapy-related toxicity comprises a braindisease, damage or malfunction. In a related aspect, the brain disease,damage or malfunction comprises CAR-T cell related NT or CAR-T cellrelated encephalopathy syndrome (CRES). In a related aspect, inhibitingor reducing incidence of a brain disease, damage or malfunctioncomprises reducing headaches, delirium, anxiety, tremor, seizureactivity, confusion, alterations in wakefulness, hallucinations,dysphasia, ataxia, apraxia, facial nerve palsy, motor weakness,seizures, nonconvulsive EEG seizures, altered levels of consciousness,coma, endothelial activation, vascular leak, intravascular coagulation,or any combination thereof in the subject. In another aspect, theimmunotherapy-related toxicity comprises CAR-T induced Cytokine ReleaseSyndrome (CRS). In a related aspect, inhibiting or reducing incidence ofCRS comprises reducing or inhibiting, without limitation, high fever,myalgia, nausea, hypotension, hypoxia, or shock, or a combinationthereof. In a related aspect, the immunotherapy-related toxicity islife-threatening.

In another aspect, the serum concentration of ANG2 or VWF, or the serumANG2:ANG1 ratio of the subject is reduced. In a related aspect, thesubject has a body temperature above 38° C., an IL-6 serumconcentration >16 pg/ml, or an MCP-1 serum concentration above 1,300pg/ml during the first 36 hours after infusion of said CAR-T cells. In arelated aspect, the subject is predisposed to have said brain disease,damage or malfunction. In a related aspect, the subject has an ANG2:ANG1ratio in serum above 1 prior to the infusion of said CAR-T cells.

In another aspect, the immunotherapy-related toxicity compriseshemophagocytic lymphohistiocytosis (HLH) or macrophage-activationsyndrome (MAS). In a related aspect, inhibiting or reducing incidence ofHLH or MAS comprises increasing survival time and/or time to relapse,reducing macrophage activation, reducing T cell activation, reducing theconcentration of IFNγ in the peripheral circulation, or reducing theconcentration of GM-CSF in the peripheral circulation, or anycombination thereof.

In another aspect, the subject presents with fever, splenomegaly,cytopenias involving two or more lines, hypertriglyceridemia,hypofibrinogenemia, hemophagocytosis, low or absent NK-cell activity,ferritin serum concentration above 500 U/ml, or soluble CD25 serumconcentration above 2400 U/ml, or any combination thereof. In a relatedaspect, the subject is predisposed to acquiring HLH or MAS. In a relatedaspect, the subject carries a mutation in a gene selected from: PRF1,UNC13D, STX11, STXBP2, or RAB27A, or has reduced expression of perforin,or any combination thereof.

In one embodiment, the GM-CSF antagonist is an anti-hGM-CSF antibody. Inanother embodiment, the anti-hGM-CSF antibody blocks binding of hGM-CSFto the alpha subunit of the hGM-CSF receptor. In another embodiment, theanti-hGM-CSF antibody is a polyclonal antibody. In another embodiment,the anti-hGM-CSF antibody is a monoclonal antibody. In anotherembodiment, the anti-hGM-CSF antibody is an antibody fragment that is aFab, a Fab′, a F(ab′)2, a scFv, or a dAB. In some embodiments, themonoclonal anti-hGM-CSF antibody, the single-chain Fv, and the Fab maybe generated in the chicken; chicken IgY are avian equivalents ofmammalian IgG antibodies. (Park et al., Biotechnology Letters (2005)27:289-295; Finley et al., Appl. Environ. Microbiol., May 2006, p.3343-3349). Chicken IgY antibodies have the following advantages: higheravidity, i.e., overall strength of binding between an antibody and anantigen, higher specificity (less cross reactivity with mammalianproteins other than the immunogen); high yield in the egg yolk, andlower background (the structural difference in the Fc region of IgY andIgG results in less false positive staining). In another embodiment, theanti-hGM-CSF antibody may be a camelid, e.g., a llama-derived singlevariable domain on a heavy chain antibodies lacking light chains (alsocalled sdAbs, VHHs and Nanobodies®); the VHH domain (about 15 kDa) isthe smallest known antigen recognition site that occurs in mammalshaving full binding capacity and affinities (equivalent to conventionalantibodies). (Garaicoechea et al. (2015) PLoS ONE 10(8): e0133665;Arbabi-Ghahroudi M (2017) Front. Immunol. 8:1589; Wu et al.,Translational Oncology (2018) 11, 366-373). In another embodiment, theantibody fragment is conjugated to polyethylene glycol. In anotherembodiment, the anti-hGM-CSF antibody has an affinity ranging from about5 pM to about 50 pM. In another embodiment, anti-hGM-CSF antibody is aneutralizing antibody. In another embodiment, the anti-hGM-CSF antibodyis a recombinant or chimeric antibody. In another embodiment, theanti-hGM-CSF antibody is a human antibody. In another embodiment, theanti-hGM-CSF antibody comprises a human variable region. In anotherembodiment, the anti-hGM-CSF antibody comprises an engineered humanvariable region. In another embodiment the anti-hGM-CSF antibodycomprises a humanized variable region. In another embodiment, theanti-hGM-CSF antibody comprises an engineered human variable region. Inanother embodiment the anti-hGM-CSF antibody comprises a humanizedvariable region.

In one embodiment, the anti-hGM-CSF antibody comprises a human lightchain constant region. In another embodiment, the anti-hGM-CSF antibodycomprises a human heavy chain constant region. In another embodiment,the human heavy chain constant region is a gamma chain. In anotherembodiment, the anti-hGM-CSF antibody binds to the same epitope aschimeric 19/2. In another embodiment, the anti-hGM-CSF antibodycomprises the VH region CDR3 and VL region CDR3 of chimeric 19/2. Inanother embodiment, the anti-GM-CSF antibody comprises the VH region andVL region CDR1, CDR2, and CDR3 of chimeric 19/2.

In one embodiment, the anti-hGM-CSF antibody comprises a heavy chainvariable region that comprises a CDR3 binding specificity determinantRQRFPY (SEQ ID NO: 12) or RDRFPY (SEQ ID NO: 13), a J segment, and aV-segment, wherein the J-segment comprises at least 95% identity tohuman JH4 (YFDYWGQGTLVTVSS (SEQ ID NO: 14)) and the V-segment comprisesat least 90% identity to a human germ line VH1 1-02 (SEQ ID NO: 19) orVH1 1-03 (SEQ ID NO:20) sequence; or a heavy chain variable region thatcomprises a CDR3 binding specificity determinant comprising RQRFPY (SEQID NO: 12). In another embodiment, the J segment comprisesYFDYWGQGTLVTVSS (SEQ ID NO: 14). In another embodiment, the CDR3comprises RQRFPYYFDY (SEQ ID NO: 15) or RDRFPYYFDY (SEQ ID NO: 16). Inanother embodiment, the heavy chain variable region CDR1 or CDR2 can bea human germline VH1 sequence; or both the CDR1 and CDR2 can be humangermline VH1. In another embodiment, the antibody comprises a heavychain variable region CDR1 or CDR2, or both CDR1 and CDR2, as shown in aV_(H) region set forth in FIG. 1. In another embodiment, theanti-hGM-CSF antibody has a V-segment that has a V_(H) V-segmentsequence shown in FIG. 1. In another embodiment, the V_(H) that has thesequence of VH #1 (SEQ ID NO:1), VH #2 (SEQ ID NO:2), VH #3 (SEQ IDNO:3), VH #4 (SEQ ID NO:4), or VH #5 (SEQ ID NO:5) set forth in FIG. 1.

In another embodiment, the anti-hGM-CSF antibody, e.g., that has a heavychain variable region as described in the paragraph above, comprises alight chain variable region that comprises a CDR3 binding specificitydeterminant comprising the amino acid sequence FNK or FNR.

In another embodiment, the anti-hGM-CSF antibody comprises a VL regionthat comprises a CDR3 comprising the amino acid sequence FNK or FNR. Inone embodiment, the anti-GM-CSF antibody comprises a human germline JK4region. In another embodiment, the antibody V_(L) region CDR3 comprisesQQFN(K/R)SPLT (SEQ ID NO: 17). In another embodiment, the anti-GM-CSFantibody comprises a VL region that comprises a CDR3 comprisingQQFNKSPLT (SEQ ID NO: 18). In another embodiment, the VL regioncomprises a CDR1, or a CDR2, or both a CDR1 and CDR2, of a V_(L) regionshown in FIG. 1. In another embodiment, the VL region comprises a Vsegment that has at least 95% identity to the VKIIIA27 (SEQ ID NO: 21)V-segment sequence as shown in FIG. 1. In another embodiment, the V_(L)region has the sequence of VK #1 (SEQ ID NO: 6), VK #2 (SEQ ID NO: 7),VK #3 (SEQ ID NO: 8), or VK #4 (SEQ ID NO: 9) set forth in FIG. 1.

In one embodiment, the anti-hGM-CSF antibody has a VH region CDR3binding specificity determinant RQRFPY (SEQ ID NO: 12) or RDRFPY (SEQ IDNO: 13) and a VL region that has a CDR3 comprising QQFNKSPLT (SEQ ID NO:18). In another embodiment, the anti-hGM-CSF antibody has a VH regionsequence set forth in FIG. 1 and a VL region sequence set forth inFIG. 1. In another embodiment, the VH region or the VL region, or boththe VH and VL region amino acid sequences comprise a methionine at theN-terminus. In another embodiment, the GM-CSF antagonist is selectedfrom the group comprising of an anti-hGM-CSF receptor antibody or asoluble GM-CSF receptor, a cytochrome b562 antibody mimetic, a hGM-CSFpeptide analog, an adnectin, a lipocalin scaffold antibody mimetic, acalixarene antibody mimetic, and an antibody like bindingpeptidomimetic.

In one embodiment, disclosed herein is a method of increasing theefficacy of CAR-T immunotherapy in a subject, the method comprising astep of administering a recombinant hGM-CSF antagonist to the subject,wherein said administering increases the efficacy of CAR-T immunotherapyin said subject. In another embodiment, said administering a recombinanthGM-CSF antagonist occurs prior to, concurrent with, or following saidCAR-T immunotherapy. In another embodiment, said increased efficacycomprises increased CAR-T cell expansion, reduced myeloid-derivedsuppressor cell (MDSC) number that inhibit T-cell function, synergy witha checkpoint inhibitor, or any combination thereof. In anotherembodiment, said increased CAR-T cell expansion comprises at least a 50%increase compared to a control. In another embodiment, said increasedCAR-T cell expansion comprises at least a one quarter log expansioncompared to a control. In another embodiment, said increased cellexpansion comprises at least a one-half log expansion compared to acontrol. In another embodiment, said increased cell expansion comprisesat least a one log expansion compared to a control. In anotherembodiment, said increased cell expansion comprises a greater than onelog expansion compared to a control.

In an embodiment, the hGM-CSF antagonist comprises a neutralizingantibody. In another embodiment, the neutralizing antibody is amonoclonal antibody.

In an embodiment, disclosed herein is a method of inhibiting or reducingthe incidence or the severity of CAR-T related toxicity in a subject,the method comprising a step of administering a recombinant hGM-CSFantagonist to the subject, wherein said administering inhibits orreduces the incidence or the severity of CAR-T related toxicity in saidsubject. In an embodiment, said CAR-T related toxicity comprises NT,CRS, or a combination thereof. In some embodiments, the CAR-T cellrelated NT is reduced by about 50% compared to a reduction in NT in asubject treated with CAR-T cells and a control antibody. In variousembodiments, the recombinant hGM-CSF antagonist is a hGM-CSFneutralizing antibody in accordance with embodiments described herein.

In another embodiment, said inhibiting or reducing incidence of CRScomprises increasing survival time and/or time to relapse, reducingmacrophage activation, reducing T cell activation, or reducing theconcentration of circulating hGM-CSF, or any combination thereof. Inanother embodiment, said subject presents with fever (with or withoutrigors, malaise, fatigue, anorexia, myalgia, arthralgia, nausea,vomiting, headache, skin rash, diarrhea, tachypnea, hypoxemia, hypoxia,shock, cardiovascular tachycardia, widened pulse pressure, hypotension,capillary leak, increased early cardiac output, diminished late cardiacoutput, elevated D-dimer, hypofibrinogenemia with or without bleeding,azotemia, transaminitis, hyperbilirubinemia, mental status changes,confusion, delirium, frank aphasia, hallucinations, tremor, dysmetria,altered gait, seizures, organ failure, or any combination thereof.

In another embodiment, the inhibiting or reducing the incidence or theseverity of CAR-T related toxicity comprises preventing the onset ofCAR-T related toxicity.

In another embodiment, disclosed herein is a method of blocking orreducing GM-CSF expression in a cell, comprising knocking out orsilencing GM-CSF gene expression in a cell. In an embodiment, theblocking or reducing of GM-CSF expression comprises short interferingRNS (siRNA), CRISPR, RNAi, DNA-directed RNA interference (ddRNAi), whichis a gene-silencing technique that uses DNA constructs to activate ananimal cell's endogenous RNA interference (RNAi) pathways, or targetedgenome editing with engineered transcription activator-like effectornucleases (TALENs), i.e., artificial proteins composed of a customizablesequence-specific DNA-binding domain fused to a nuclease that cleavesDNA in a nonsequence-specific manner. (Joung and Sander, Nat Rev MolCell Biol. 2013 January; 14(1): 49-55), which is incorporated herein inits entirety by reference. In an embodiment, the cell is a CAR-T cell.

In one embodiment, the subject is a human.

In one embodiment, disclosed herein is a hGM-CSF antagonist for use in amethod of inhibiting or reducing the incidence or severity ofimmunotherapy-related toxicity in a subject, the method comprising astep of administering a recombinant hGM-CSF antagonist to the subject.In one embodiment, disclosed herein is a pharmaceutical compositioncomprising an anti-hGM-CSF antagonist.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides exemplary V_(H) and V_(L) sequences of anti-GM-CSFantibodies.

FIGS. 2A-2B illustrates binding of GM-CSF to Ab1 (FIG. 2A) or Ab2 (FIG.2B) determined by surface plasmon resonance analysis at 37° C. (Biacore3000). Ab1 and Ab2 were captured on anti Fab polyclonal antibodiesimmobilized on the Biacore chip. Different concentrations of GM-CSF wereinjected over the surface as indicated. Global fit analysis was carriedout assuming a 1:1 interaction using Scrubber2 software.

FIGS. 3A-3B illustrates binding of Ab1 and Ab2 to glycosylated (FIG. 3A)and non-glycosylated GM-CSF (FIG. 3B). Binding to glycosylated GM-CSFexpressed from human 293 cells or non-glycosylated GM-CSF expressed inE. coli was determined by ELISA. Representative results from a singleexperiment are shown (exp 1). Two-fold dilutions of Ab1 and Ab2 startingfrom 1500 ng/ml were applied to GM-CSF coated wells. Each pointrepresents mean±standard error for triplicate determinations. Sigmoidalcurve fit was performed using Prism 5.0 Software (Graphpad).

FIGS. 4A-4B illustrates competition ELISA demonstrating binding of Ab1and Ab2 to a shared epitope. ELISA plates coated with 50 ng/well ofrecombinant GM-CSF were incubated with various concentrations ofantibody (Ab2, Ab1 or isotype control antibody) together with 50 nMbiotinylated Ab2. Biotinylated antibody binding was assayed usingneutravidin-HRP conjugate. Competition for binding to GM-CSF was for 1hr (FIG. 4A) or for 18 hrs (FIG. 4B). Each point representsmean±standard error for triplicate determinations. Sigmoidal curve fitwas performed using Prism 5.0 Software (Graphpad).

FIG. 5 illustrates inhibition of GM-CSF-induced IL-8 expression. Variousamounts of each antibody were incubated with 0.5 ng/ml GM-CSF andincubated with U937 cells for 16 hrs. IL-8 secreted into the culturesupernatant was determined by ELISA.

FIG. 6 illustrates dose-dependent inhibition of GM-CSF-stimulated CD11bon human granulocytes by anti-GM-CSF antibody.

FIG. 7 illustrates dose-dependent inhibition of GM-CSF-induced HLA-DR onCD14+ human, primary monocytes/macrophages by anti-GM-CSF antibody.

FIG. 8 illustrates the role of GM-CSF (Myeloid Inflammatory Factor) as akey cytokine in CAR-T-related activity and in stimulation of white bloodcell proliferation, which is a characteristic feature in certainleukemias, e.g., acute myeloid leukemia (AML).

FIG. 9 illustrates inhibition of GM-CSF-dependent human TF-1 cellproliferation (human erythroleukemia) by neutralization of human GM-CSFwith anti-GM-CSF antibody. KB003 is a recombinant monoclonal antibodydesigned to target and neutralize human GM-CSF. KB002 is a mouse/humanchimeric monoclonal antibody, that targets and neutralizes hGM-CSF.

FIG. 10 is a depiction of a chimeric antigen receptor.

FIG. 11 illustrates CAR-T19 therapy results in high response rates inrelapsed refractory ALL. Data show historic outcomes in R/R ALL andoutcomes in R/R ALL after CAR-T19 therapy. (Maude, et al NEJM 2014).

FIG. 12 illustrates evidence showing a significant GM-CSF link to NT.GM-CSF levels correlate with serious adverse effects after CAR-T celltherapy. GM-CSF levels precede and modulate other cytokines other thanIL-15. Elevated GM-CSF is clearly associated with ≥grade 3 NT. IL-2 isonly other cytokine with this association.

FIG. 13 illustrates an estimated time course of CRS and NT followingCD19 CAR-T cell therapy. Timing of symptom onset and CRS severitydepends on the inducing agent, type of cancer, age of patient, and themagnitude of immune cell activation. CAR-T related CRS symptom onsettypically occurs days to occasionally weeks after the T-cell infusion,coinciding with maximal T-cell expansion. Similar to CRS associated withmAb therapy, CRS associated with adoptive T-cell therapies has beenconsistently associated with elevated IFNγ, IL-6, TNFα, IL-1, IL-2,IL-6, GM-CSF, IL-10, IL-8, and IL-5. No clear CAR-T cell dose-responserelationship for CRS exists, but very high doses of T cells may resultin earlier onset of symptoms.

FIG. 14 illustrates that GM-CSF is a key initiator of CAR-T adverseeffects. The figure depicts the central role of GM-CSF in CRS and NT.Perforin allows granzymes to penetrate the tumor cell membrane. CAR-Tproduced GM-CSF recruits CCR2+ myeloid cells to the tumor site, whichproduce CCL2 (MCP1). CCL2 positively reinforces its own production byCCR2+ myeloid cell recruitment. IL-1 and IL-6 from myeloid cells formanother positive feedback loop with CAR-T by inducing production ofGM-CSF. Phosphatidyl serine is exposed as a result of perforin andgranzyme cell membrane destruction. Phosphatidyl-serine stimulatesmyeloid cell production of CCL2, IL-1, IL-6, and other inflammatoryeffectors. The final outcome of this self-reinforcing feedback loopresults in endothelial activation, vascular permeability, andultimately, CRS and NT. Moreover, animal model evidence shows GM-CSFknockout mice show no sign of CRS, but IL-6 knockout mice can stilldevelop CRS. GM-CSF receptor k/o from CCR2+myeloid cells abrogatescascade in neuro-inflammation models. (Sentman, et al., J. Immunol.;Coxford, et al. Immunity 2015 (43)510-514; Ishii et al., Blood 2016128:3358; Teachey, et al. Cancer Discov. 2016 Jun. 6(6): 664-679; Lee,et al., Blood 2016 124:2:188; Barrett, et al., Blood 2016: 128-654, eachof which is incorporated in its entirety herein by reference.).

FIGS. 15A-15G illustrate that GM-CSF CRISPR knockout T-cells exhibitreduced expression of GM-CSF but similar levels of other cytokines anddegranulation. a. Generation of GM-CSF knockout CAR-Ts. (See Example 6).

FIGS. 16A-16J illustrate that GM-CSF neutralizing antibody in accordancewith embodiments described herein does not inhibit CAR-T mediatedkilling, proliferation, or cytokine production but successfullyneutralizes GM-CSF (See Example 7).

FIGS. 17A-17B illustrate the protocol and results from a mouse model ofhuman CRS. (Example 5).

FIGS. 18A-18C illustrate CAR-T efficacy in a xenograft model incombination with a GM-CSF neutralizing antibody in accordance withembodiments described herein. The GM-CSF neutralizing antibody is shownto not inhibit CAR-T efficacy in vivo. (See Example 8).

FIG. 19 illustrates in vitro and in vivo preclinical data showing that aGM-CSF neutralizing antibody in accordance with embodiments describedherein did not impair CAR-T impact on survival. The GM-CSF neutralizingantibody does not impede CAR-T cell function in vivo in the absence ofPBMCs. Survival was similar for CAR-T+control and CAR-T+GM-CSFneutralizing antibody. (See Example 9).

FIGS. 20A-20B illustrate in vitro and in vivo preclinical data showingthat a GM-CSF neutralizing antibody in accordance with embodimentsdescribed herein does increase CAR-T expansion. The GM-CSF neutralizingantibody increases in vitro CAR-T cancer cell killing. Antibodyneutralization of GM-CSF increases proliferation of CAR-T cells in thepresence of PBMCs. CAR-T proliferation increased by the GM-CSFneutralizing antibody in presence of PBMCs. (It was not affected withoutPBMCs). The anti-GM-CSF antibody did not inhibit CAR-T degranulation,intracellular GM-CSF production, or IL-2 production. (See Example 10).

FIG. 21 illustrates that CAR-T expansion is associated with improvedoverall response rate. CAR AUC (area under the curve) defined ascumulative levels of CAR+cells/μL of blood over the first 28 days postCAR-T administration. P values calculated by Wilcoxon rank sum test.(Neelapu, et al ICML 2017 Abstract 8). (See Example 11).

FIG. 22 illustrates a study protocol for GM-CSF neutralizing antibody inaccordance with embodiments described herein. (See Example 12). CRS andNT to be assessed daily while hospitalized and at clinic visit for first30 days. Eligible subjects to receive GM-CSF neutralizing antibody ondays −1, +1, and +3 of CAR-T treatment. Additional dosing can becontemplated going out to at least day 7. Tumor assessment to beperformed at baseline and months 1, 3, 6, 9, 12, 18, and 24. Bloodsamples (PBMC and serum) days −5, −1, 0, 1, 3, 5, 7, 9, 11, 13, 21, 28,90, 180, 270, and 360. (See Example 12).

FIGS. 23A-23B illustrate that GM-CSF depletion increases CAR-T cellexpansion. FIG. 23A illustrates an increased ex-vivo expansion ofGM-CSF^(k/o) CAR-T cells compared to control CAR-T cells. FIG. 23Billustrates a more robust CAR-T cell proliferation after treatment witha GM-CSF neutralizing antibody in accordance with embodiments describedherein. (See Example 13).

FIG. 24 illustrates a safety profile of GM-CSF neutralizing antibody inaccordance with embodiments described herein. (See Example 14).

FIGS. 25A-25D illustrate that GM-CSF neutralizing antibody when added toCAR-T cell therapy demonstrates a 90% reduction in neuroinflammation inmouse preclinical model. FIG. 25A illustrates MRI data (T1hyperintensity indicative of BBB disruption and neuroinflammation) inwhich mice brains are protected from neuroinflammation afteradministration of CAR-T cells and GM-CSF neutralizing antibody inaccordance with embodiments described herein compared to mice brainsshowing signs of neurotoxicity after administration of CAR-T cells and acontrol antibody (top row) and compared to untreated (baseline) micebrains (bottom row). FIG. 25B quantitatively illustrates the percentincrease of T1 hyperintensity from baseline: there was an approximately10% percent increase in brain T1 hyperintensity from baseline in miceadministered CAR-T and GM-CSF neutralizing antibody in accordance withembodiments described herein compared to the slightly over 100% increasein mice that had been administered CAR-T cells and control antibody. Asshown in the comparative graph, the ˜10% increase in brain T1hyperintensity from baseline in mice administered the CAR-T and GM-CSFneutralizing antibody is a 90% reduction in neuroinflammation, asmeasured by brain T1 hyperintensity from baseline, compared to thequantity of neuroinflammation present in mice that received CAR-T cellsand control antibody. FIGS. 25C-25D show that compared to untreated mice(which had 500,000 to 1.5M leukemic cells) and CAR-T plus controlantibody (which had between 15,000 and 100,000 leukemic cells),treatment with CAR-T plus GM-CSF neutralizing antibody in accordancewith embodiments described herein led to a significant reduction in thenumber of leukemic cells (decreased to between 500 and 5,000 cells) withimproved overall disease control (See Example 15).

FIGS. 26A-26I show that GM-CSF blockade helps control CART19 toxicitiesand does improve efficacy. FIG. 26A shows CART19 and lenzilumab treatedCART19 are equally effective in survival outcomes in a high tumor burdenNALM6 relapse model compared to UTD (untransduced T cells) (7-8 mice pergroup, n=2). FIGS. 26B-26D show Lenzilumab & anti-mouse GM-CSFantibody-controlled CRS induced weight loss, neutralized serum humanGM-CSF, and reduced expression of serum mouse MCP-1 (monocytechemoattractant protein-1) in a primary ALL xenograft CART19 CRS/NTmodel (3 mice per group, * p<0.05). FIG. 26E shows Lenzilumab &anti-mouse GM-CSF antibody reduced brain inflammation as shown by MRI ina primary ALL xenograft CART19 CRS/NT model (3 mice per group, * p<0.05,** p<0.01). FIGS. 26F-26G show an improved efficacy of CART19+Lenzilumabtreated mice compared to anti-mouse GM-CSF antibody treated mice, i.e.,CART19+anti-hGM-CSF antibody, showed reduced CD19+ brain leukemic burdenand reduced percentage of brain macrophages in a primary ALL xenograftCART19 CRS/NT model (3 mice per group). FIG. 26H shows CRISPR Cas9 K/Oof GM-CSF reduces its expression via intracellular staining in CART19and UTD with NALM6 stimulation. (Representative experiment, n=2) FIG.26I shows CART19 and GM-CSF K/O CART19 control tumor burden better thanUTD, and an improved efficacy of GM/CSF K/O CART19 cells controllingtumor burden slightly better than CART19 in a high tumor burden NALM6relapse model (6 mice per group, * p<0.05, **** p<0.0001). Error barsSEM.

FIGS. 27A-27D show GM-CSF neutralization in vitro enhances CAR-T cellproliferation in the presence of monocytes and does not impair CAR-Tcell effector function. FIG. 27A graphically depicts Lenzilumab (ahGM-CSF neutralizing antibody) neutralization of CAR-T cell producedhGM-CSF in vitro compared to isotype control treatment as assayed bymultiplex after 3 days of culture with CART19 in media alone or CART19co-cultured with NALM6, n=2 experiments, 2 replicates per experiment,representative experiment depicted, *** p<0.001 between lenzilumab andisotype control treatment, t test, mean+SEM. FIG. 27B graphically showsthat hGM-CSF neutralizing antibody treatment did not inhibit the abilityof CAR-T cells to proliferate as assayed by CSFE flow cytometryproliferation assay of live CD3 cells, n=3 donors, 2 replicates perdonor, representative experiment at 3-day time point depicted, ns p>0.05between lenzilumab and isotype control treatment, t test, mean+SEM.Alone: CART19 in media alone, MOLM13: CART19+MOLM13, PMA/ION: CART19plus 5 ng/mL PMA and 0.1 ug/mL ION, NALM6: CART19+NALM6. FIG. 27Cgraphically depicts Lenzilumab enhancing the proliferation of CART19 byneutralization of hGM-CSF compared to isotype control treated withCART19 when co-cultured with human monocytes, n=3 donors at 3-day timepoint, 2 replicates per donor, **** p<0.0001, mean+SEM. FIG. 27Dgraphically shows that Lenzilumab treatment did not inhibit cytotoxicityof CART19 or untransduced T cells (UTD) when cultured with NALM6, n=3donors, 2 replicates per donor, representative experiment at 48 hr timepoint depicted, ns p>0.05 between lenzilumab and isotype controltreatment, t test, mean+SEM.

FIGS. 28A-28F demonstrate that GM-CSF neutralization in vivo enhancesCAR-T cell anti-tumor activity (i.e., tumor cell killing) in xenograftmodels. FIG. 28 A illustrates the experimental schema: NSG mice wereinjected with the CD19+ luciferase+ cell line NALM6 (1×106 cells permouse I.V). 4-6 days later, mice were imaged, randomized, and received1-1.5×106CAR-T19 or equivalent number of total cells of control UTDcells the following day with either lenzilumab or control IgG (10 mg/Kg,given IP daily for 10 days, starting on the day of CAR-T injection).Mice were followed with serial bioluminescence imaging to assess diseaseburden beginning day 7 post CAR-T cell injection and were followed foroverall survival. Tail vein bleeding was performed 7-8 days after CAR-Tcell injection. FIG. 28B depicts Lenzilumab neutralization of CAR-Tproduced serum hGMCSF in vivo compared to isotype control treatment asassayed by hGM-CSF singleplex, n=2 experiments, 7-8 mice per group,representative experiment, serum from day 8 post CAR-T cell/UTDinjection, *** p<0.001 between lenzilumab and isotype control treatment,t test, mean+SEM. FIG. 28C graphically depicts Lenzilumab treated CAR-Tin vivo are equally effective at controlling tumor burden compared toisotype control treated CAR-T in a high tumor burden relapse xenograftmodel of ALL, day 7 post CAR-T injection, n=2 experiments, 7-8 mice pergroup, representative experiment depicted, *** p<0.001, * p<0.05, nsp>0.05, t test, mean+SEM. FIG. 28 D depicts mouse images from FIG. 28C.FIG. 28E illustrates the experimental schema: NSG mice were injectedwith the blasts derived from patients with ALL (1×106 cells per mouseI.V). Mice were bled serially and when the CD19+ cells >1/uL, mice wererandomized to receive 2.5×106 CART19 with either lenzilumab or controlIgG (10 mg/Kg, given IP daily for 10 days, starting on the day of CAR-Tinjection). Mice were followed with serial tail vein bleeding to assessdisease burden beginning day 14 post CAR-T cell injection and werefollowed for overall survival. FIG. 28F graphically depicts thatLenzilumab treatment with CAR-T therapy results in more sustainedcontrol of tumor burden over time in a primary acute lymphoblasticleukemia (ALL) xenograft model compared to isotype control treatmentwith CAR-T therapy, 6 mice per group, ** p<0.01, * p<0.05, ns p>0.05, ttest, mean+SEM.

FIGS. 29A-29E demonstrate that GM-CSF CRISPR knockout CAR-T cellsexhibit reduced expression of GM-CSF, similar levels of key cytokinesand chemokines, and enhanced anti-tumor activity. FIG. 29A illustratesthat the CRISPR Cas9 GM-CSF^(k/o) CART19 exhibit reduced GM-CSFproduction compared to wild type CART19, but other cytokine productionand degranulation are not inhibited by the GM-CSF gene disruption,CART19 and GM-CSF^(k/o) CART19 stimulated with NALM6, n=3 experiments, 2replicates per experiment, *** p<0.001, * p<0.05, ns p>0.05 comparingGM-CSF^(k/o) CART19 and CAR19, t test, mean+SEM. FIG. 29B illustratesthat GM-CSF^(k/o) CAR-T have reduced serum human GM-CSF in vivo comparedto CAR-T treatment as assayed by multiplex, 5-6 mice per group (4-6 attime of bleed, 8 days post CAR-T cell injection), **** p<0.0001, ***p<0.001 between GM-CSF^(k/o) CART19 and wild type CART19, t test,mean+SEM. FIG. 29C illustrates that GM-CSF^(k/o) CART19 in vivo enhancesoverall survival compared to wild type CART19 in a high tumor burdenrelapse xenograft model of ALL utilizing a NALM6 cell line, 5-6 mice pergroup, ** p<0.01, log-rank. FIGS. 29D-29E show human (FIG. 29D) andmouse (FIG. 29E) cytokines and chemokines from multiplex of serum, otherthan hGM-CSF, show no statistical differences between the GM-CSF^(k/o)CART19 and wild type CART19, further implicating critical T-cellcytokines and chemokines are not adversely depleted by reducing GM-CSFexpression, 5-6 mice per group (4-6 at time of bleed), **** p<0.0001, ttest.

FIGS. 30A-30D illustrate a patient derived xenograft model forneuro-inflammation and cytokine release syndrome. FIG. 30A shows theexperimental schema: Mice received 1-3×106 primary blasts derived fromthe peripheral blood of patients with primary ALL. Mice were monitoredfor engraftment for ≥10-13 weeks via tail vein bleeding. When serumCD19+ cells were >10 cells/uL, the mice received CART19 (2-5×106 cells)and commenced antibody therapy for a total of 10 days, as indicated.Mice were weighed on a daily basis as a measure of their wellbeing.Mouse brain MRIs were performed 5-6 days post CART19 injection and tailvein bleeding for cytokine/chemokine and T cell analysis was performed4-11 days post CART19 injection, 2 independent experiments. FIG. 30Billustrates that a combination of GM-CSF neutralization with CART19 isequally effective as isotype control antibodies combined with CART19 incontrolling CD19+ burden of ALL cells, representative experiment, 3 miceper group, 11 days post CART19 injection, * p<0.05 between GM-CSFneutralization+CART19 and isotype control+CART19, t test, mean+SEM. FIG.30C illustrates brain MRI data showing CART19 therapy exhibits T1enhancement, suggestive of brain blood-brain barrier disruption andpossible edema. 3 mice per group, 5-6 days post CART19 injection,representative image. FIG. 30D illustrates high tumor burden primary ALLxenografts treated with CART19 show human CD3 cell infiltration of thebrain compared to untreated PDX controls. 3 mice per group,representative image.

FIG. 31 shows the canonical pathways altered in brains from patientderived xenografts after treatment with CART19 cells. Red boxes indicateupregulation of genes in CART19 plus isotype control treated micecompared to the untreated patient derived xenografts.

FIGS. 32A-32D demonstrate GM-CSF neutralization in vivo ameliorates CRSafter CART19 therapy in a xenograft model. FIG. 32A shows Lenzilumab andanti-mouse GM-CSF antibody prevent CRS induced weight loss compared tomice treated with CART19 and isotype control antibodies, 3 mice pergroup, 2-way anova, mean+SEM. FIG. 32B shows human GM-CSF wasneutralized in patient derived xenografts treated with lenzilumab andmouse GM-CSF neutralizing antibody, 3 mice per group, *** p<0.001, *p<0.05, t test, mean+SEM. FIG. 32C shows human cytokine/chemokine heatmap (serum collected 11 days after CART19 injection) exhibits increasesin cytokines and chemokines typical of CRS after CART19 treatment. GMCSFneutralization results in a significant decrease in several cytokinesand chemokines compared to mice treated with CART19 and isotype controlantibodies, including several myeloid associated cytokines andchemokines, as indicated in the panel, 3 mice per group, serum from day11 post CART19 injection, *** p<0.001, ** p<0.01, * p<0.05, comparingGM-CSF neutralizing antibody treated and isotype control treated micethat received CAR-T cell therapy, t test. FIG. 32D shows mousecytokine/chemokine heat map (serum collected 11 days after CART19injection) exhibit increase in mouse cytokines and chemokines typical ofCRS after CART19 treatment. GM-CSF neutralization results in asignificant decrease in several cytokines and chemokines compared totreatment with CART19 with control antibodies, including several myeloiddifferentiating cytokines and chemokines, as indicated in the panel, 3mice per group, serum from day 11 post CART19 injection, * p<0.05,comparing GM-CSF neutralizing antibody treated and isotype controltreated mice that received CAR-T cell therapy, t test.

FIGS. 33A-33D demonstrate GM-CSF neutralization in vivo amelioratesneuro-inflammation after CART19 therapy in a xenograft model. FIGS.33A-33B depict gadolinium enhanced T1-hyperintensity (cubic mm) MRIshowed that GM-CSF neutralization helped reduced brain inflammation,blood-brain barrier disruption, and possible edema compared to isotypecontrol (A) representative images, (33B) 3 mice per group, ** p<0.01, *p<0.05, 1-way ANOVA, mean+SD. FIG. 33C shows human CD3 T cells werepresent in the brain after treatment with CART19 therapy. GM-CSFneutralization resulted in a trend toward decreased CD3 infiltration inthe brain as assayed by flow cytometry in brain hemispheres, 3 mice pergroup, mean+SEM. FIG. 33D depicts CD11b+ bright macrophages weredecreased in the brains of mice receiving GM-CSF neutralization duringCAR-T therapy compared to isotype control during CAR-T therapy asassayed by flow cytometry in brain hemispheres, 3 mice per group,mean+SEM.

FIGS. 34A(i)-34B illustrate the generation of GM-CSF^(k/o) CART19 cells.FIGS. 34A(i)-34A(iv) show the experimental schema; FIG. 34B shows thegRNA sequence and primer sequences for generation of GM-CSF^(k/o)CART19. To generate GM-CSF^(k/o) CART19 cells, gRNA was cloned into aCas9 lentivirus vector under the control of a U6 promotor and used forlentivirus production. T cells derived from normal donors werestimulated with CD3/CD28 beads and dual transduced with CAR19 virus andCRISPR/Cas9 virus 24 hours later. CD3/CD28 magnetic bead removal wasperformed on Day +6, and GM-CSF^(k/o) CART19 cells or control CART19cells were cryopreserved on Day 8.

FIG. 35 shows a flow chart for procedures used in RNA sequencing. Thebinary base call data was converted to fastq using Illumina bcl2fastqsoftware. The adapter sequences were removed using Trimmomatic, andFastQC was used to check for quality. The latest human (GRCh38) andmouse (GRCm38) reference genomes were downloaded from NCBI. Genome indexfiles were generated using STAR, and the paired end reads were mapped tothe genome for each condition. HTSeq was used to generate expressioncounts for each gene, and DeSeq2 was used to calculate differentialexpression. Gene ontology was assessed using Enrichr.

FIG. 36 shows that Lenzilumab plus CAR-T cell treated mice havecomparable survival compared to isotype control antibody plus CAR-T celltreated mice in a high tumor burden relapse xenograft model of ALL. n=2experiments, 7-8 mice per group, representative experiment depicted,**** p<0.0001, *** p<0.001, * p<0.05, log-rank.

FIG. 37 shows a representative TIDE sequence to verify genome alterationin the GM-CSF CRISPR Cas9 knockout CAR-T cells. n=2 experiments,representative experiment depicted.

FIG. 38 shows GM-CSF knockout CAR-T cells in vivo show slightly enhancedcontrol of tumor burden compared to wild type CAR-T cells in a hightumor burden relapse xenograft model of ALL. Days post CAR-T cellinjection listed on x-axis, 5-6 mice per group (2 remained in UTD groupat day 13), representative experiment depicted, **** p<0.0001, * p<0.05,2-way ANOVA, mean+SEM.

FIG. 39 demonstrates a patient derived xenograft model forneuro-inflammation and CRS with CART19+ anti-hGM-CSF antibody treatment.High tumor burden primary ALL xenografts treated CART19+anti-hGM-CSFantibody treatment show human CD3 cell infiltration of the brain (FIG.39) compared to untreated PDX controls (FIG. 30D). 3 mice per group,representative image.

DETAILED DESCRIPTION OF THE INVENTION

The present subject matter may be understood more readily by referenceto the following detailed description which forms a part of thisdisclosure. It is to be understood that this disclosure is not limitedto the specific products, methods, conditions or parameters describedand/or shown herein, and that the terminology used herein is for thepurpose of describing particular embodiments by way of example only andis not intended to be limiting of the claimed disclosure.

Immunotherapy-Related Toxicity

A skilled artisan would appreciate that the term “immunotherapy-relatedtoxicity” refers to a spectrum of inflammatory symptoms resulting fromhigh levels of immune activation. Different types of toxicity areassociated with different immunotherapy approaches. In some embodiments,immunotherapy-related toxicity comprises capillary leak syndrome,cardiac disease, respiratory disease, CAR-T-cell-related encephalopathysyndrome (CRES), neurotoxicity, colitis, convulsions, cytokine releasesyndrome (CRS), cytokine storm, decreased left ventricular ejectionfraction, diarrhea, disseminated intravascular coagulation, edema,encephalopathy, exanthema, gastrointestinal bleeding, gastrointestinalperforation, hemophagocytic lymphohistiocytosis (HLH), hepatosis,hypertension, hypophysitis, immune related adverse events,immunohepatitis, immunodeficiencies, ischemia, liver toxicity,macrophage-activation syndrome (MAS), pleural effusions, pericardialeffusions, pneumonitis, polyarthritis, posterior reversibleencephalopathy syndrome (PRES), pulmonary hypertension, thromboembolism,and transaminitis.

While different types of toxicities differ in their pathophysiology andclinical manifestations, they are usually associated with an increase ininflammation-associated factors, such as C-reactive protein, GM-CSF,IL-1, IL-2, sIL-2Rα, IL-5, IL-6, IL-8, IL-10, IP10, IL-15, MCP-1 (AKACCL2), MIG, MIP-1β, IFNγ, CX3CR1, or TNFα. A skilled artisan wouldappreciate that, in some embodiments, the term “inflammation-associatedfactor” comprises molecules, small molecules, peptides, genetranscripts, oligonucleotides, proteins, hormones, and biomarkers thatare affected during inflammation. A skilled artisan would appreciatethat systems affected during inflammation comprises upregulation,downregulation, activation, de-activation, or any kind of molecularmodification. The serum concentration of inflammation-associatedfactors, such as cytokines, can be used as an indicator ofimmunotherapy-related toxicities, and may be expressed as—fold increase,percent (%) increase, net increase or rate of change in cytokine levelsor concentration. The concentration of inflammation-associated factorsin body fluids other than serum can also be used as indicators ofimmunotherapy-related toxicities. In some embodiments, absolute cytokinelevels or concentrations above a certain level or concentration may bean indication of a subject undergoing or about to experience animmunotherapy-related toxicity. In another embodiment, absolute cytokinelevels or concentration at a certain level, for example a level orconcentration normally found in a control subject, may be an indicationof a method for inhibiting or reducing the incidence of animmunotherapy-related toxicity in a subject. A skilled artisan wouldappreciate that the term “cytokine level” may encompass a measure ofconcentration, a measure of fold change, a measure of percent (%)change, or a measure of rate change. Further, the methods for measuringcytokines in blood, cerebrospinal fluid (CSF), saliva, serum, urine, andplasma are well known in the art.

A number of approaches have been elaborated to classify the type ofneurotoxicity and manage it accordingly. These classifications are basedon clinical and biological symptoms, as fever, hypotension, hypoxia,organ toxicity, cardiac dysfunction, respiratory dysfunction,gastrointestinal dysfunction, hepatic dysfunction, renal dysfunction,coagulopathy, seizure presence, intracranial pressure, muscle tone,motor performance, ferritin levels, and haemagophagocytosis. Similarly,each type of neurotoxicity can be graded according to its severity.Table 1A (taken from Cellular Therapy Implementation: the MDACCApproach, P. Kebriaei, Feb. 24, 2017) discloses a method for gradingneurotoxicity according to its severity into Grade 1, Grade 2, Grade 3,and Grade 4. However, some of the foregoing symptoms are not typicallyassociated with neurotoxicity. (Lee, et al., Blood 2014; 124:188-195,which is incorporated in its entirety herein by reference.).

TABLE 1A Method for Grading Neurotoxicity - Criteria for Adverse Events(CTCAE) Symptom or sign Grade 1 Grade 2 Grade 3 Grade 4 Level of MildModerate somnolence, Obtundation or stupor Life-threateningconsciousness drowsiness/ limiting instrumental needing urgentsleepiness ADL intervention or mechanical ventilation Orientation/ MildModerate Severe disorientation, Life-threatening Confusiondisorientation/ disorientation, limiting self-care ADL needing urgentconfusion limiting instrumental intervention or ADL mechanicalventilation ADL/ Mild limiting Limiting instrumental Limiting self-careLife-threatening Encephalopathy of ADL ADL ADL needing urgentintervention or mechanical ventilation Speech Dysphasia Dysphasia withSevere receptive or — not impairing moderate impairment expressivedysphasia, ability to in ability to impairing ability to communicatecommunicate read, write or spontaneously communicate intelligiblySeizure Brief partial Brief generalized Multiple seizuresLife-threatening; seizure; no seizure despite medical prolonged loss ofintervention repetitive seizures consciousness Incontinent or motorBowel/bladder weakness incontinence; Weakness limiting selfcare ADL,disabling MD Mild (7-9) Moderate (3-6) Severe (1-2), grade 1 Critical(Obtunded; Anderson Cancer and 2 papilledema convulsive status Center(MDACC) with CSF opening epilepticus; motor 10-point pressure (op) <20mmHg weakness, grade 3, Neurotoxicity grade 4 & 5 papilledema, CSF op≥20 mmHg, cerebral edema)

Patients with body temperature above 38.9° C., IL-6 serum concentrationabove 16 pg/ml, or MCP-1 (AKA CCL2) serum concentration above 1,343.5pg/ml in the first 36 hours after immunotherapy infusion had higherprobabilities of developing severe neurotoxicity (Gust, et al. CancerDiscov. 2017 Oct. 12).

CRS is a serious condition and life-threatening adverse effect becauseof abnormal cytokine regulation and thus, severe inflammation. Symptomscan include, without limitation, fever, disordered heartbeat andbreathing, nausea, vomiting, and seizures. CRS can be graded byassessing symptoms and their severities, such as, for example: Grade 1CRS: Fever, constitutional symptoms; Grade 2 CRS: Hypotension—respondsto fluids or one low dose pressor, Hypoxia—responds to <40% O₂, Organtoxicity; grade 2; Grade 3 CRS: Hypotension—requires multiple pressorsor high dose pressors, Hypoxia—requires ≥40% O₂, Organ toxicity—grade 3,grade 4 transaminitis; Grade 4 CRS: Mechanical ventilation, Organtoxicity—grade 4, excluding transaminitis. (Lee, et al., Blood 2014;124:188-195, which is incorporated in its entirety herein byreference.).

CRES can be graded, for example, by combining neurological assessmentwith other parameters as papilloedema, CSF opening pressure, imagingassessment, and the presence of seizures and motor weakness. A methodfor grading CRES is described in Neelapu et al., Nat Rev Clin Oncol.15(1):47-62 (2018) (Epub 2017 Sep. 19), which is incorporated in itsentirety herein by reference. Table 1B (taken from Neelapu et al., NatRev Clin Oncol. 15(1):47-62 (2018)) discloses a method for grading CRESaccording to its severity into Grade 1, Grade 2, Grade 3, and Grade 4.

TABLE 1B Method for grading CRES. In CARTOX-10, a point is assigned foreach of the following tasks performed correctly: orientation to year,month, city, hospital, and President/Prime Minister of country ofresidence (1 point for each); naming three objects (1 point for each);writing a standard sentence; counting backwards from 100 in tens.Symptom or sign Grade 1 Grade 2 Grade 3 Grade 4 Neurological 7-9 (mild3-6 0-2 (severe Patient in critical condition, assessment scoreimpairment) (moderate impairment) and/or (by CARTOX-10) impairment)obtunded and cannot perform assessment of tasks Raised intracranial NANA Stage 1-2 Stage 3-5 papilloedema, or pressure papilloedema, or CSFopening CSF pressure ≥20 mmHg, or opening pressure cerebral oedema <20mmHg Seizures or motor NA NA Partial seizure, or Generalized seizures,or weakness non-convulsive convulsive or seizures on EEG non-convulsivestatus with response to epilepticus, or new benzodiazepine motorweakness

NT, CRS, and CRES manifestations can include encephalopathy, headaches,delirium, anxiety, tremor, seizure activity, confusion, alterations inwakefulness, decreased level of consciousness, hallucinations,dysphasia, aphasia, ataxia, apraxia, facial nerve palsy, motor weakness,seizures, nonconvulsive EEG seizures, cerebral edema, and coma. CRES isassociated with elevated concentrations of circulating cytokines, asC-reactive protein, GM-CSF, IL-1, IL-2, sIL2Rα, IL-5, IL-6, IL-8, IL-10,IP10, IL-15, MCP-1, MIG, MIP1β, IFNγ, CX3CR1, and TNFα.

The cytokine concentration gradient between serum and CSF observed innormal conditions is reduced or lost during CRES. Additionally, CART-cells and high protein concentrations are observed in the CSF ofpatients and is correlated with the severity of the condition. All thisindicates a blood-brain barrier dysfunction following immunotherapy.Increased vascular permeability can be partially explained by increasedconcentrations of ANG2 and increased ANG2:ANG1 ratio in patients withneurotoxicity. While ANG1 induces endothelial cell quiescence, ANG2causes endothelial cell activation and microvascular permeability.Patients with increased endothelial activation before immunotherapy werereported to have higher probability of suffering neurotoxicity (Gust, etal. Cancer Discov. 2017 Oct. 12).

Hemophagocytic lymphohistiocytosis (HLH) comprises severehyperinflammation caused by uncontrolled proliferation of benignlymphocytes and macrophages that secrete high amounts of inflammatorycytokines. In some embodiments, HLH can be classified as one of thecytokine storm syndromes. In some embodiments, HLH occurs after strongimmunologic activation, such as systemic infections, immunodeficiency,malignancies. or immunotherapy. In some embodiments, the term “HLH” maybe used interchangeably with the terms “hemophagocyticlymphohistiocytosis”, “hemophagocytic syndrome”, or “hemophagocyticsyndrome” having all the same qualities and meanings.

Primary HLH comprises a heterogeneous autosomal recessive disorder.Patients with homozygous mutations in one of several genes, exhibit lossof function of proteins involved in cytolytic granule exocytosis. Insome embodiments, HLH can present in infancy with minimal or no trigger.Secondary HLH, or acquired HLH, occurs after strong immunologicactivation, such as that which occurs with systemic infection,immunodeficiency, an underlying malignancy, or immunotherapies. Bothforms of HLH are characterized by an overwhelming activation of normal Tlymphocytes and macrophages, invariably leading to clinical andhaematologic alterations and death in the absence of treatment.

In some embodiments, HLH can be initiated by viral infections, EBV, CMV,parvovirus, HSV, VZV, HHV8, HIV, influenza, hepatitis A, hepatitis B,hepatitis C, bacterial infections, gram-negative rods, Mycoplasmaspecies and Mycobacterium tuberculosis, parasitic infections, Plasmodiumspecies, Leishmania species, Toxoplasma species, fungal infections,Cryptococcal species, Candidal species and Pneumocystis species, amongothers.

Macrophage-activation syndrome (MAS) comprises a condition comprisinguncontrolled activation and proliferation of macrophages, and Tlymphocytes, with a marked increase in circulating cytokine levels, suchas IFNγ, and GM-CSF. MAS is closely related to secondary HLH. MASmanifestations include high fever, hepatosplenomegaly, lymphadenopathy,pancytopenia, liver dysfunction, disseminated intravascular coagulation,hemophagocytosis, hypofibrinogenemia, hyperferritinemia, andhypertriglyceridemia.

CRS comprises a non-antigen-specific immune response similar to thatfound in severe infection. CRS is characterized by any or all of thefollowing symptoms: fever with or without rigors, malaise, fatigue,anorexia, myalgias, arthalgias, nausea, vomiting, headache, skin rash,diarrhea, tachypnea, hypoxemia, hypoxia, shock, cardiovasculartachycardia, widened pulse pressure, hypotension, capillary leak,increased cardiac output (early), potentially diminished cardiac output(late), elevated D-dimer, hypofibrinogenemia with or without bleeding,azotemia, transaminitis, hyperbilirubinemia, headache, mental statuschanges, confusion, delirium, word finding difficulty or frank aphasia,hallucinations, tremor, dysmetria, altered gait, seizures, organfailure, multi-organ failure. Deaths have also been reported. Severe CRShas been reported to occur in up to 60% of patients receiving CAR-T19.

Cytokine storm comprises an immune reaction consisting of a positivefeedback loop between cytokines and white blood cells, with highlyelevated levels of various cytokines. The term “cytokine storm” may beused interchangeably with the terms “cytokine cascade” and“hypercytokinemia” having all the same qualities and meanings. In someembodiments, a cytokine storm is characterized by IL-2 release andlymphoproliferation. Cytokine storm leads to potentiallylife-threatening complications including cardiac dysfunction, adultrespiratory distress syndrome, neurologic toxicity, renal and/or hepaticfailure, and disseminated intravascular coagulation.

As noted, CAR-T cell therapy is currently limited by the risk oflife-threatening neurotoxicity and CRS. Despite active management, allCAR-T responders experience some degree of CRS. Up to 50% of patientstreated with CD19 CAR-T have at least Grade 3 CRS or neurotoxicity.GM-CSF levels and T-cell expansion are the factors most associated withgrade 3 or higher CRS and neurotoxicity.

Reducing or eliminating CRS and neurotoxicity in immunotherapies such asCAR-T cell therapy is of great value and it is crucial to determine whatis driving or exacerbating the signature CAR-T inflammatory response.Although many cytokines, signaling molecules, and cell types areinvolved in this pathway, GM-CSF is the one cytokine that appears to beat the center of the pathway. Normally undetectable in human serum, itis central to the cyclical positive feedback loop that drivesinflammation to the extremes of cytokine storms and endothelial cellactivation. Neurotoxicity and cytokine storms are not the result of asimultaneous release of cytokines, but rather a cascade of inflammationinitiated by GM-CSF resulting in the trafficking and recruitment ofmyeloid cells to the tumor site. These myeloid cells produce thecytokines observed in CRS and neurotoxicity, perpetuating theinflammatory cascade.

Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF)

As used herein, “Granulocyte Macrophage-Colony Stimulating Factor”(GM-CSF) refers to a small, naturally occurring glycoprotein withinternal disulfide bonds having a molecular weight of approximately 23kDa. In some embodiments, GM-CSF refers to human GM-CSF. In someembodiments, GM-CSF refers to non-human GM-CSF. In humans, it is encodedby a gene located within the cytokine cluster on human chromosome 5. Thesequence of the human gene and protein are known. The protein has anN-terminal signal sequence, and a C-terminal receptor binding domain(Rasko and Gough In: The Cytokine Handbook, A. Thomson, et al, AcademicPress, New York (1994) pages 349-369). Its three-dimensional structureis similar to that of the interleukins, although the amino acidsequences are not similar GM-CSF is produced in response to a number ofinflammatory mediators by mesenchymal cells present in the hemopoieticenvironment and at peripheral sites of inflammation. GM-CSF is able tostimulate the production of neutrophilic granulocytes, macrophages, andmixed granulocyte-macrophage colonies from bone marrow cells and canstimulate the formation of eosinophil colonies from fetal liverprogenitor cells. GM-CSF can also stimulate some functional activitiesin mature granulocytes and macrophages. GM-CSF, a cytokine present inthe bone marrow microenvironment, recruits inflammatory monocyte-deriveddendritic cells, stimulates the secretion of high levels of IL-6 andCCL2/MCP-1, and leads to a feedback loop, recruiting more monocytes,inflammatory dendritic cells to inflammatory sites.

As noted, CRS involves the increase of several cytokines and chemokines,including IFN-γ, IL-6, IL-8, CCL2 (MCP-1), CCL3 (MIP1a), and GM-CSF.(Teachey, D. et al. (June 2016), Cancer Discovery, CD-16-0040; MorganR., et al., (April 2010), Molecular Therapy.). IL-6, one of the keyinflammatory cytokines, is not produced by CAR-T cells. (Barrett, D. etal. (2016), Blood). Instead, it is produced by myeloid cells, which arerecruited to the tumor site. GM-CSF mediates this recruitment, whichinduces chemokine production that activates myeloid cells and causesthem to traffic to the tumor site. Elevated GM-CSF levels serve as botha predictive biomarker for CRS and an indicator of its severity. Morethan a critical component of the inflammation cascade, GM-CSF is the keyinitiator, responsible for both CRS and NT. As described herein, in vivostudies using murine models indicate that genetic silencing of GM-CSFprevents cytokine storm—while still maintaining CAR-T efficacy. GM-CSFknockout mice have normal levels of INF-γ, IL-6, IL-10, CCL2 (MCP1),CCL3/4 (MIG-1) in vivo and do not develop CRS. (Sentman, M.-L., et al(2016), The Journal of Immunology, 197(12), 4674-4685.). GM-CSF knockoutCAR-T models recruit fewer NK cells, CD8 cells, myeloid cells, andneutrophils to the tumor site in comparison to GM-CSF+ CAR-T.

The term “soluble granulocyte macrophage-colony stimulating factorreceptor” (sGM-CSFR) refers to a non-membrane bound receptor that bindsGM-CSF, but does not transduce a signal when bound to the ligand.

As used herein, a “peptide GM-CSF antagonist” refers to a peptide thatinteracts with GM-CSF, or its receptor, to reduce or block (eitherpartially or completely) signal transduction that would otherwise resultfrom the binding of GM-CSF to its cognate receptor expressed on cells.GM-CSF antagonists may act by reducing the amount of GM-CSF ligandavailable to bind the receptor (e.g., antibodies that once bound toGM-CSF increase the clearance rate of GM-CSF) or prevent the ligand frombinding to its receptor either by binding to GM-CSF or the receptor(e.g., neutralizing antibodies). GM-CSF antagonists may also includeother peptide inhibitors, which may include polypeptides that bindGM-CSF or its receptor to partially or completely inhibit signaling. Apeptide GM-CSF antagonist can be, e.g., an antibody; a natural orsynthetic GM-CSF receptor ligand that antagonizes GM-CSF, or otherpolypeptides. An exemplary assay to detect GM-CSF antagonist activity isprovided in Example 1. Typically, a peptide GM-CSF antagonist, such as aneutralizing antibody, has an EC50 of 10 nM or less.

A “purified” GM-CSF antagonist as used herein refers to a GM-CSFantagonist that is substantially or essentially free from componentsthat normally accompany it as found in its native state. For example, aGM-CSF antagonist such as an anti-GM-CSF antibody that is purified fromblood or plasma is substantially free of other blood or plasmacomponents such as other immunoglobulin molecules. Purity andhomogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis orhigh-performance liquid chromatography. A protein that is thepredominant species present in a preparation is substantially purified.Typically, “purified” means that the protein is at least 85% pure, morepreferably at least 95% pure, and most preferably at least 99% purerelative to the components with which the protein naturally occurs.

Antibodies

As used herein, an “antibody” refers to a protein functionally definedas a binding protein and structurally defined as comprising an aminoacid sequence that is recognized by one of skill as being derived fromthe framework region of an immunoglobulin-encoding gene of an animalthat produces antibodies. An antibody can consist of one or morepolypeptides substantially encoded by immunoglobulin genes or fragmentsof immunoglobulin genes. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon and mu constant regiongenes, as well as myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

A typical immunoglobulin (antibody) structural unit is known to comprisea tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain (VL)and variable heavy chain (V_(H)) refer to these light and heavy chains,respectively.

The term “antibody” includes antibody fragments that retain bindingspecificity. For example, there are a number of well characterizedantibody fragments. Thus, for example, pepsin digests an antibodyC-terminal to the disulfide linkages in the hinge region to produceF(ab′)2, a dimer of Fab which itself is a light chain joined to VH-CH1by a disulfide bond. The F(ab′)2 may be reduced under mild conditions tobreak the disulfide linkage in the hinge region thereby converting the(Fab′)2 dimer into a Fab′ monomer. The Fab′ monomer is essentially a Fabwith part of the hinge region (see, Fundamental Immunology, W. E. Paul,ed., Raven Press, N.Y. (1993), for a more detailed description of otherantibody fragments). While various antibody fragments are defined interms of the digestion of an intact antibody, one of skill willappreciate that fragments can be synthesized de novo either chemicallyor by utilizing recombinant DNA methodology. Thus, the term antibody, asused herein also includes antibody fragments either produced by themodification of whole antibodies or synthesized using recombinant DNAmethodologies.

Antibodies include dimers such as V_(H)-V_(L) dimers, V_(H) dimers, orV_(L) dimers, including single chain antibodies (antibodies that existas a single polypeptide chain), such as single chain Fv antibodies (sFvor scFv), in which a variable heavy and a variable light region arejoined together (directly or through a peptide linker) to form acontinuous polypeptide. The single chain Fv antibody is a covalentlylinked V_(H)-V_(L) heterodimer which may be expressed from a nucleicacid including V_(H)- and V_(L)-encoding sequences either joineddirectly or joined by a peptide-encoding linker (e.g., Huston, et al.Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). While the V_(H) andV_(L) are connected to each as a single polypeptide chain, the V_(H) andV_(L) domains associate non-covalently. Alternatively, the antibody canbe another fragment, such as a disulfide-stabilized Fv (dsFv). Otherfragments can also be generated, including using recombinant techniques.The scFv antibodies and a number of other structures converting thenaturally aggregated, but chemically separated light and heavypolypeptide chains from an antibody V region into a molecule that foldsinto a three-dimensional structure substantially similar to thestructure of an antigen-binding site and are known to those of skill inthe art (see e.g., U.S. Pat. Nos. 5,091,513, 5,132,405, and 4,956,778).In some embodiments, antibodies include those that have been displayedon phage or generated by recombinant technology using vectors where thechains are secreted as soluble proteins, e.g., scFv, Fv, Fab, (Fab′)2 orgenerated by recombinant technology using vectors where the chains aresecreted as soluble proteins. Antibodies for use in the invention canalso include diantibodies and miniantibodies.

Antibodies of the invention also include heavy chain dimers, such asantibodies from camelids. Since the V_(H) region of a heavy chain dimerIgG in a camelid does not have to make hydrophobic interactions with alight chain, the region in the heavy chain that normally contacts alight chain is changed to hydrophilic amino acid residues in a camelid.V_(H) domains of heavy-chain dimer IgGs are called VHH domains.Antibodies for use in the current invention include single domainantibodies (dAbs) and nanobodies (see, e.g., Cortez-Retamozo, et al.,Cancer Res. 64:2853-2857, 2004).

As used herein, “V-region” refers to an antibody variable region domaincomprising the segments of Framework 1, CDR1, Framework 2, CDR2, andFramework 3, including CDR3 and Framework 4, which segments are added tothe V-segment as a consequence of rearrangement of the heavy chain andlight chain V-region genes during B-cell differentiation. A “V-segment”as used herein refers to the region of the V-region (heavy or lightchain) that is encoded by a V gene. The V-segment of the heavy chainvariable region encodes FR1-CDR1-FR2-CDR2 and FR3. For the purposes ofthis invention, the V-segment of the light chain variable region isdefined as extending though FR3 up to CDR3.

As used herein, the term “J-segment” refers to a subsequence of thevariable region encoded comprising a C-terminal portion of a CDR3 andthe FR4. An endogenous J-segment is encoded by an immunoglobulin J-gene.

As used herein, “complementarity-determining region (CDR)” refers to thethree hypervariable regions in each chain that interrupt the four“framework” regions established by the light and heavy chain variableregions. The CDRs are primarily responsible for binding to an epitope ofan antigen. The CDRs of each chain are typically referred to as CDR1,CDR2, and CDR3, numbered sequentially starting from the N-terminus, andare also typically identified by the chain in which the particular CDRis located. Thus, for example, a V_(H) CDR3 is located in the variabledomain of the heavy chain of the antibody in which it is found, whereasa V_(L) CDR1 is the CDR1 from the variable domain of the light chain ofthe antibody in which it is found.

The sequences of the framework regions of different light or heavychains are relatively conserved within a species. The framework regionof an antibody, that is the combined framework regions of theconstituent light and heavy chains, serves to position and align theCDRs in three-dimensional space.

The amino acid sequences of the CDRs and framework regions can bedetermined using various well-known definitions in the art, e.g., Kabat,Chothia, international ImMunoGeneTics database (IMGT), and AbM (see,e.g., Johnson et al., supra; Chothia & Lesk, 1987, Canonical structuresfor the hypervariable regions of immunoglobulins. J. Mol. Biol. 196,901-917; Chothia C. et al., 1989, Conformations of immunoglobulinhypervariable regions. Nature 342, 877-883; Chothia C. et al., 1992,structural repertoire of the human VH segments J. Mol. Biol. 227,799-817; Al-Lazikani et al., J. Mol. Biol 1997, 273(4)). Definitions ofantigen combining sites are also described in the following: Ruiz etal., IMGT, the international ImMunoGeneTics database. Nucleic AcidsRes., 28, 219-221 (2000); and Lefranc, M.-P. IMGT, the internationalImMunoGeneTics database. Nucleic Acids Res. January 1; 29(1):207-9(2001); MacCallum et al, Antibody-antigen interactions: Contact analysisand binding site topography, J. Mol. Biol., 262 (5), 732-745 (1996); andMartin et al, Proc. Natl Acad. Sci. USA, 86, 9268-9272 (1989); Martin,et al, Methods Enzymol., 203, 121-153, (1991); Pedersen et al,Immunomethods, 1, 126, (1992); and Rees et al, In Sternberg M. J. E.(ed.), Protein Structure Prediction. Oxford University Press, Oxford,141-172 1996).

“Epitope” or “antigenic determinant” refers to a site on an antigen towhich an antibody binds. Epitopes can be formed both from contiguousamino acids or noncontiguous amino acids juxtaposed by tertiary foldingof a protein. Epitopes formed from contiguous amino acids are typicallyretained on exposure to denaturing solvents whereas epitopes formed bytertiary folding are typically lost on treatment with denaturingsolvents. An epitope typically includes at least 3, and more usually, atleast 5 or 8-10 amino acids in a unique spatial conformation. Methods ofdetermining spatial conformation of epitopes include, for example, x-raycrystallography and 2-dimensional nuclear magnetic resonance. See, e.g.,Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66,Glenn E. Morris, Ed (1996).

The term “binding specificity determinant” or “BSD” as used in thecontext of the current invention refers to the minimum contiguous ornon-contiguous amino acid sequence within a CDR region necessary fordetermining the binding specificity of an antibody. In the currentinvention, the minimum binding specificity determinants reside within aportion or the full-length of the CDR3 sequences of the heavy and lightchains of the antibody.

As used herein, “anti-GM-CSF antibody” or “GM-CSF antibody” are usedinterchangeably to refer to an antibody that binds to GM-CSF andinhibits GM-CSF receptor binding and activation. Such antibodies may beidentified using any number of art-recognized assays that assess GM-CSFbinding and/or function. For example, binding assays such as ELISAassays that measure the inhibition of GM-CSF binding to the alphareceptor subunit may be used. Cell-based assays for GM-CSF receptorsignaling, such as assays which determine the rate of proliferation of aGM-CSF-dependent cell line in response to a limiting amount of GM-CSF,are also conveniently employed, as are assays that measure amounts ofcytokine production, e.g., IL-8 production, in response to GM-CSFexposure.

As used herein, “neutralizing antibody” refers to an antibody that bindsto GM-CSF and inhibits signaling by the GM-CSF receptor, or preventsbinding of GM-CSF to its receptor.

As used herein, “human Granulocyte Macrophage-Colony Stimulating Factor”(hGM-CSF) refers to a small naturally occurring glycoprotein withinternal disulfide bonds having a molecular weight of approximately 23kDa; the source and the target of the GM-CSF are human; as such,anti-hGM-CSF antibody, as described in embodiments herein, binds onlyhuman and primate GM-CSF, but not mouse, rat, and other mammalianGM-CSF. The hGM-CSF antibodies, as described in embodiments herein,neutralize human GM-CSF. In some embodiments, the hGM-CSF in humans isencoded by a gene located within the cytokine cluster on humanchromosome 5. The sequences of the human gene and protein are known. Theprotein has an N-terminal signal sequence, and a C-terminal receptorbinding domain (Rasko and Gough In: The Cytokine Handbook, A. Thomson,et al., Academic Press, New York (1994) pages 349-369). Itsthree-dimensional structure is similar to that of the interleukins,although the amino acid sequences are not similar. GM-CSF is produced inresponse to a number of inflammatory mediators present in thehemopoietic environment and at peripheral sites of inflammation. GM-CSFis able to stimulate the production of neutrophilic granulocytes,macrophages, and mixed granulocyte-macrophage colonies from bone marrowcells and can stimulate the formation of eosinophil colonies from fetalliver progenitor cells. GM-CSF can also stimulate some functionalactivities in mature granulocytes and macrophages and inhibits apoptosisof granulocytes and macrophages.

The term “equilibrium dissociation constant” or “affinity” abbreviated(K_(D)), refers to the dissociation rate constant (k_(d), time⁻¹)divided by the association rate constant (k_(a), time⁻¹ M⁻¹).Equilibrium dissociation constants can be measured using any knownmethod in the art. The antibodies of the present invention are highaffinity antibodies. Such antibodies have a monovalent affinity better(less) than about 10 nM, and often better than about 500 pM or betterthan about 50 pM as determined by surface plasmon resonance analysisperformed at 37° C. Thus, in some embodiments, the antibodies of theinvention have an affinity (as measured using surface plasmonresonance), of less than 50 pM, typically less than about 25 pM, or evenless than 10 pM.

In some embodiments, an anti-GM-CSF antibody of the invention has a slowdissociation rate with a dissociation rate constant (kd) determined bysurface plasmon resonance analysis at 37° C. for the monovalentinteraction with GM-CSF less than approximately 10⁻⁴ s⁻¹, preferablyless than 5×10⁻⁵ s⁻¹ and most preferably less than 10⁻⁵ s⁻¹.

As used herein, “chimeric antibody” refers to an immunoglobulin moleculein which (a) the constant region, or a portion thereof, is altered,replaced or exchanged so that the antigen binding site (variable region)is linked to a constant region of a different or altered class, effectorfunction and/or species, or an entirely different molecule that confersnew properties to the chimeric antibody, e.g., an enzyme, toxin,hormone, growth factor, drug, etc.; or (b) the variable region, or aportion thereof, is altered, replaced or exchanged with a variableregion, or portion thereof, having a different or altered antigenspecificity; or with corresponding sequences from another species orfrom another antibody class or subclass.

As used herein, “humanized antibody” refers to an immunoglobulinmolecule in CDRs from a donor antibody are grafted onto human frameworksequences. Humanized antibodies may also comprise residues of donororigin in the framework sequences. The humanized antibody can alsocomprise at least a portion of a human immunoglobulin constant region.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. Humanization can be performed using methods known in the art(e.g., Jones et al., Nature 321:522-525; 1986; Riechmann et al., Nature332:323-327, 1988; Verhoeyen et al., Science 239:1534-1536, 1988);Presta, Curr. Op. Struct. Biol. 2:593-596, 1992; U.S. Pat. No.4,816,567), including techniques such as “superhumanizing” antibodies(Tan et al., J. Immunol. 169: 1119, 2002) and “resurfacing” (e.g.,Staelens et al., Mol. Immunol. 43: 1243, 2006; and Roguska et al., Proc.Natl. Acad. Sci USA 91: 969, 1994).

A “HUMANEERED®” antibody in the context of this invention refers to anengineered human antibody having a binding specificity of a referenceantibody. An engineered human antibody for use in this invention has animmunoglobulin molecule that contains minimal sequence derived from adonor immunoglobulin. In some embodiments, the engineered human antibodymay retain only the minimal essential binding specificity determinantfrom the CDR3 regions of a reference antibody. Typically, an engineeredhuman antibody is engineered by joining a DNA sequence encoding abinding specificity determinant (BSD) from the CDR3 region of the heavychain of the reference antibody to human V_(H) segment sequence and alight chain CDR3 BSD from the reference antibody to a human V_(L)segment sequence. A “BSD” refers to a CDR3-FR4 region, or a portion ofthis region that mediates binding specificity. A binding specificitydeterminant therefore can be a CDR3-FR4, a CDR3, a minimal essentialbinding specificity determinant of a CDR3 (which refers to any regionsmaller than the CDR3 that confers binding specificity when present inthe V region of an antibody), the D segment (with regard to a heavychain region), or other regions of CDR3-FR4 that confer the bindingspecificity of a reference antibody. Methods for engineering humanantibodies are provided in US patent application publication no.20050255552 and US patent application publication no. 20060134098.

The term “human antibody” as used herein refers to an antibody that issubstantially human, i.e., has FR regions, and often CDR regions, from ahuman immune system. Accordingly, the term includes humanized andhumaneered antibodies as well as antibodies isolated from micereconstituted with a human immune system and antibodies isolated fromdisplay libraries.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not normally found in the same relationship toeach other in nature. For instance, the nucleic acid is typicallyrecombinantly produced, having two or more sequences, e.g., fromunrelated genes arranged to make a new functional nucleic acid.Similarly, a heterologous protein will often refer to two or moresubsequences that are not found in the same relationship to each otherin nature.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, e.g., recombinant cells express genes that are not foundwithin the native (non-recombinant) form of the cell or express nativegenes that are otherwise abnormally expressed, under-expressed or notexpressed at all. By the term “recombinant nucleic acid” herein is meantnucleic acid, originally formed in vitro, in general, by themanipulation of nucleic acid, e.g., using polymerases and endonucleases,in a form not normally found in nature. In this manner, operable linkageof different sequences is achieved. Thus, an isolated nucleic acid, in alinear form, or an expression vector formed in vitro by ligating DNAmolecules that are not normally joined, are both considered recombinantfor the purposes of this invention. It is understood that once arecombinant nucleic acid is made and reintroduced into a host cell ororganism, it will replicate non-recombinantly, i.e., using the in vivocellular machinery of the host cell rather than in vitro manipulations;however, such nucleic acids, once produced recombinantly, althoughsubsequently replicated non-recombinantly, are still consideredrecombinant for the purposes of the invention. Similarly, a “recombinantprotein” is a protein made using recombinant techniques, i.e., throughthe expression of a recombinant nucleic acid.

The phrase “specifically (or selectively) binds” to an antibody or is“specifically (or selectively) immunoreactive with”, refers to a bindingreaction where the antibody binds to the antigen of interest. In thecontext of this invention, the antibody typically binds to the antigen,e.g., GM-CSF, with an affinity of 500 nM or less, and has an affinity of5000 nM or greater, for other antigens.

The terms “identical” or percent “identity,” in the context of two ormore polypeptide (or nucleic acid) sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues (or nucleotides) that are the same(i.e., about 60% identity, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specifiedregion, when compared and aligned for maximum correspondence over acomparison window or designated region) as measured using a BLAST orBLAST 2.0 sequence comparison algorithms with default parametersdescribed below, or by manual alignment and visual inspection (see,e.g., NCBI web site). Such sequences are then said to be “substantiallyidentical.” “Substantially identical” sequences also includes sequencesthat have deletions and/or additions, as well as those that havesubstitutions, as well as naturally occurring, e.g., polymorphic orallelic variants, and man-made variants. As described below, thepreferred algorithms can account for gaps and the like. Preferably,protein sequence identity exists over a region that is at least about 25amino acids in length, or more preferably over a region that is 50-100amino acids=in length, or over the length of a protein.

A “comparison window”, as used herein, includes reference to a segmentof one of the number of contiguous positions selected from the groupconsisting typically of from 20 to 600, usually about 50 to about 200,more usually about 100 to about 150 in which a sequence may be comparedto a reference sequence of the same number of contiguous positions afterthe two sequences are optimally aligned. Methods of alignment ofsequences for comparison are well-known in the art. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by thehomology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443(1970), by the search for similarity method of Pearson & Lipman, Proc.Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Dr.,Madison, Wis.), or by manual alignment and visual inspection (see, e.g.,Current Protocols in Molecular Biology (Ausubel et al., eds. 1995supplement)).

An indication that two polypeptides are substantially identical is thatthe first polypeptide is immunologically cross reactive with theantibodies raised against the second polypeptide. Thus, a polypeptide istypically substantially identical to a second polypeptide, e.g., wherethe two peptides differ only by conservative substitutions.

Preferred examples of algorithms that are suitable for determiningpercent sequence identity and sequence similarity include the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nuc. AcidsRes. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410(1990). BLAST and BLAST 2.0 are used, with the parameters describedherein, to determine percent sequence identity for the nucleic acids andproteins of the invention. The BLASTN program (for nucleotide sequences)uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5,N=−4 and a comparison of both strands. For amino acid sequences, theBLASTP program uses as defaults a wordlength of 3, and expectation (E)of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc.Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation(E) of 10, M=5, N=−4, and a comparison of both strands.

The terms “isolated,” “purified,” or “biologically pure” refer tomaterial that is substantially or essentially free from components thatnormally accompany it as found in its native state. Purity andhomogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis orhigh-performance liquid chromatography. A protein that is thepredominant species present in a preparation is substantially purified.The term “purified” in some embodiments denotes that a protein givesrise to essentially one band in an electrophoretic gel. Preferably, itmeans that the protein is at least 85% pure, more preferably at least95% pure, and most preferably at least 99% pure.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers, those containing modified residues, and non-naturallyoccurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction similarly to the naturally occurring amino acids. Naturallyoccurring amino acids are those encoded by the genetic code, as well asthose amino acids that are later modified, e.g., hydroxyproline,γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers tocompounds that have the same basic chemical structure as a naturallyoccurring amino acid, e.g., an a carbon that is bound to a hydrogen, acarboxyl group, an amino group, and an R group, e.g., homoserine,norleucine, methionine sulfoxide, methionine methyl sulfonium. Suchanalogs may have modified R groups (e.g., norleucine) or modifiedpeptide backbones, but retain the same basic chemical structure as anaturally occurring amino acid. Amino acid mimetics refers to chemicalcompounds that have a structure that is different from the generalchemical structure of an amino acid, but that functions similarly to anaturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refer to those nucleic acidswhich encode identical or essentially identical amino acid sequences, orwhere the nucleic acid does not encode an amino acid sequence, toessentially identical or associated, e.g., naturally contiguous,sequences. Because of the degeneracy of the genetic code, a large numberof functionally identical nucleic acids encode most proteins. Forinstance, the codons GCA, GCC, GCG and GCU all encode the amino acidalanine. Thus, at every position where an alanine is specified by acodon, the codon can be altered to another of the corresponding codonsdescribed without altering the encoded polypeptide. Such nucleic acidvariations are “silent variations,” which are one species ofconservatively modified variations. Every nucleic acid sequence hereinwhich encodes a polypeptide also describes silent variations of thenucleic acid. One of skill will recognize that in certain contexts eachcodon in a nucleic acid (except AUG, which is ordinarily the only codonfor methionine, and TGG, which is ordinarily the only codon fortryptophan) can be modified to yield a functionally identical molecule.Accordingly, often silent variations of a nucleic acid which encodes apolypeptide is implicit in a described sequence with respect to theexpression product, but not with respect to actual probe sequences.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables and substitution matrices such asBLOSUM providing functionally similar amino acids are well known in theart. Such conservatively modified variants are in addition to and do notexclude polymorphic variants, interspecies homologs, and alleles of theinvention. Typical conservative substitutions for one anotherinclude: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamicacid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S),Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g.,Creighton, Proteins (1984)).

Methods for Preventing or Treating an Immunotherapy-Related Toxicity

In some embodiments, disclosed herein are methods of inhibitingimmunotherapy-related toxicity in a subject. In some embodiments, hereinare methods of reducing the incidence of immunotherapy-related toxicityin a subject. In some embodiments, disclosed herein are methods ofneutralizing hGM-CSF. In some embodiment, the methods comprise a step ofadministering a recombinant hGM-CSF antagonist to the subject. In someembodiments, the method comprises hGM-CSF gene silencing. In someembodiments, the method comprises hGM-CSF gene knockout. Methods of genesilencing and gene knockout are well known to those of ordinary skill inthe art, and may include, without limitation, RNA interference (RNAi),CRISPR, short interfering RNS (siRNA), DNA-directed RNA interference(ddRNAi), targeted genome editing with engineered transcriptionactivator-like effector nucleases (TALENs) or other suitable techniques.

In some embodiments, inhibiting or reducing the incidence or theseverity of immunotherapy-related toxicity comprises reducing immuneactivation. In some embodiments, inhibiting or reducing the incidence orthe severity of immunotherapy-related toxicity comprises amelioratingcapillary leak syndrome. In some embodiments, inhibiting or reducing theincidence or the severity of immunotherapy-related toxicity comprisesameliorating a cardiac dysfunction. In some embodiments, inhibiting orreducing the incidence or the severity of immunotherapy-related toxicitycomprises ameliorating encephalopathy. In some embodiments, inhibitingor reducing the incidence or the severity of immunotherapy-relatedtoxicity comprises alleviating colitis. In some embodiments, inhibitingor reducing the incidence or the severity of immunotherapy-relatedtoxicity comprises inhibiting convulsions. In some embodiments,inhibiting or reducing the incidence or the severity ofimmunotherapy-related toxicity comprises ameliorating CRS. In someembodiments, inhibiting or reducing the incidence or the severity ofimmunotherapy-related toxicity comprises ameliorating neurotoxicity. Invarious embodiments, the CAR-T cell related neurotoxicity in a subjectis reduced by about 90% compared to a reduction in neurotoxicity in asubject treated with CAR-T cells and a control antibody. In certainembodiments, the recombinant GM-CSF antagonist is an antibody, inparticular, a GM-CSF neutralizing antibody in accordance withembodiments described herein, including Example 15.

In some embodiments, inhibiting or reducing the incidence or theseverity of immunotherapy-related toxicity comprises reducing cytokinestorm symptoms. In some embodiments, inhibiting or reducing theincidence or the severity of immunotherapy-related toxicity comprisesincreasing impaired left ventricular ejection fraction. In someembodiments, inhibiting or reducing the incidence or the severity ofimmunotherapy-related toxicity comprises ameliorating diarrhea. In someembodiments, inhibiting or reducing the incidence or the severity ofimmunotherapy-related toxicity comprises ameliorating disseminatedintravascular coagulation.

In some embodiments, inhibiting or reducing the incidence or theseverity of immunotherapy-related toxicity comprises reducing edema. Insome embodiments, inhibiting or reducing the incidence or the severityof immunotherapy-related toxicity comprises alleviating exanthema. Insome embodiments, inhibiting or reducing the incidence or the severityof immunotherapy-related toxicity comprises reducing gastrointestinalbleeding. In some embodiments, inhibiting or reducing the incidence orthe severity of immunotherapy-related toxicity comprises treating agastrointestinal perforation. In some embodiments, inhibiting orreducing the incidence or the severity of immunotherapy-related toxicitycomprises treating hemophagocytic lymphohistiocytosis (HLH). In someembodiments, inhibiting or reducing the incidence or the severity ofimmunotherapy-related toxicity comprises treating hepatosis. In someembodiments, inhibiting or reducing the incidence or the severity ofimmunotherapy-related toxicity comprises reducing hypotension. In someembodiments, inhibiting or reducing the incidence or the severity ofimmunotherapy-related toxicity comprises reducing hypophysitis.

In some embodiments, inhibiting or reducing the incidence or theseverity of immunotherapy-related toxicity comprises inhibiting immunerelated adverse events. In some embodiments, inhibiting or reducing theincidence or the severity of immunotherapy-related toxicity comprisesreducing immunohepatitis. In some embodiments, inhibiting or reducingthe incidence or the severity of immunotherapy-related toxicitycomprises reducing immunodeficiencies. In some embodiments, inhibitingor reducing the incidence or the severity of immunotherapy-relatedtoxicity comprises treating ischemia. In some embodiments, inhibiting orreducing the incidence or the severity of immunotherapy-related toxicitycomprises reducing liver toxicity. In some embodiments, inhibiting orreducing the incidence or the severity of immunotherapy-related toxicitycomprises treating macrophage-activation syndrome (MAS). In someembodiments, inhibiting or reducing the incidence or the severity ofimmunotherapy-related toxicity comprises reducing neurotoxicitysymptoms.

In some embodiments, inhibiting or reducing the incidence or theseverity of immunotherapy-related toxicity comprises reducing pleuraleffusions. In some embodiments, inhibiting or reducing the incidence orthe severity of immunotherapy-related toxicity comprises reducingpericardial effusions. In some embodiments, inhibiting or reducing theincidence or the severity of immunotherapy-related toxicity comprisesreducing pneumonitis.

In some embodiments, inhibiting or reducing the incidence or theseverity of immunotherapy-related toxicity comprises reducingpolyarthritis. In some embodiments, inhibiting or reducing the incidenceor the severity of immunotherapy-related toxicity comprises treatingposterior reversible encephalopathy syndrome (PRES). In someembodiments, inhibiting or reducing the incidence or the severity ofimmunotherapy-related toxicity comprises reducing pulmonaryhypertension. In some embodiments, inhibiting or reducing the incidenceor the severity of immunotherapy-related toxicity comprises treatingthromboembolism. In some embodiments, inhibiting or reducing theincidence or the severity of immunotherapy-related toxicity comprisesreducing transaminitis. In some embodiments, inhibiting or reducing theincidence or the severity of immunotherapy-related toxicity comprisesreducing a patient's CRES, neurotoxicity (NT), and/or cytokine releasesyndrome (CRS) grade. In some embodiments, inhibiting or reducing theincidence or the severity of immunotherapy-related toxicity comprisesimproving a patient's CARTOX-10 score.

In one aspect, this invention further provides a method for treating orpreventing immunotherapy-related toxicity in a subject, the methodcomprising administering to the subject chimeric antigenreceptor-expressing T-cells (CAR-T cells), the CAR-T cells having aGM-CSF gene knockout (GM-CSF^(k/o) CAR-T cells), and a recombinanthGM-CSF antagonist, as demonstrated in Examples 6 and 20-21. In someembodiments, the GM-CSF^(k/o) CAR-T cells express a reduced level ofGM-CSF compared to a level of GM-CSF expression by wild-type CAR-Tcells. In certain embodiments, the GM-CSF^(k/o) CAR-T cells express alevel of one or more cytokine and/or chemokine that is lower than orequivalent to a level of the one or more cytokine and/or chemokineexpressed by wild-type CAR-T cells. In particular embodiments, the oneor more cytokine is a human cytokine selected from the group consistingof IFN-γ, GRO, MDC, IL-2, IL-3, IL-5, IL-7, IP-10, CD107a, TNF-α andVEGF. In some embodiments the one or more cytokine is selected from thegroup consisting of IFN-γ, IL-1a, IL-1b, IL-2, IL-4, IL-5, IL-6, IL7,IL-9, IL-10, IL-12p40, IL-12p70, ILF, IL-13, LIX, IL-15, IP-10, KC,MCP-1, MIP-1a, MIP-1b, M-CSF MIP-2, MIG, RANTES, and TNF-a, eotaxin,G-CSF and a combination thereof. In various embodiments, the recombinantGM-CSF antagonist is an hGM-CSF antagonist. In some embodiments, therecombinant GM-CSF antagonist is an anti-GM-CSF antibody. In particularembodiments, the anti-GM-CSF antibody binds a human GM-CSF. In otherembodiments, the anti-GM-CSF antibody binds a primate GM-CSF. In variousembodiments, the anti-GM-CSF antibody binds a mammalian GM-CSF. In someembodiments, the anti-GM-CSF antibody is an anti-hGM-CSF antibody. Incertain embodiments, the anti-hGM-CSF antibody is a monoclonal antibody.In various embodiments, the anti-hGM-CSF antibody is an antibodyfragment that is a Fab, a Fab′, a F(ab′)2, a scFv, or a dAB. In someembodiments, the anti-hGM-CSF antibody is a human GM-CSF neutralizingantibody. In certain embodiments, the anti-hGM-CSF antibody is arecombinant or chimeric antibody. In various embodiments, theanti-hGM-CSF antibody is a human antibody. In some embodiments, theCAR-T cells are CD19 CAR-T cells. In particular embodiments, theGM-CSF^(k/o) CAR-T cells enhance anti-tumor activity of the recombinanthGM-CSF antagonist. In specific embodiments, the GM-CSF^(k/o) CAR-Tcells improve overall survival of the subject compared to survival in asubject treated by administration of wild-type CAR-T cells. Inparticular embodiments, administering to the subject the CAR-T cellshaving a GM-CSF gene knockout (GM-CSF^(k/o) CAR-T cells) and arecombinant hGM-CSF antagonist is a durable treatment for preventing ortreating an immunotherapy-related toxicity, such as CRS, neurotoxicityand neuroinflammation. In some embodiments, the subject has cancer. Invarious embodiments, the cancer is acute lymphoblastic leukemia.

Methods for Reducing Relapse Rate or Preventing Occurrence

In one aspect, this invention provides a method for reducing relapserate or preventing occurrence of tumor relapse in a subject treated withimmunotherapy, the method comprising administering to the subject arecombinant GM-CSF antagonist. In some embodiments, the reducing relapserate or preventing occurrence of tumor relapse in the subject occurs inan absence of an incidence of immunotherapy-related toxicity. In certainembodiments, the reducing relapse rate or preventing occurrence of tumorrelapse in the subject occurs in a presence of an incidence ofimmunotherapy-related toxicity. In some embodiments, the recombinantGM-CSF antagonist is an hGM-CSF antagonist. In various embodiments, therecombinant GM-CSF antagonist is an anti-GM-CSF antibody. In someembodiments, the anti-GM-CSF antibody binds a human GM-CSF. In certainembodiments, the anti-GM-CSF antibody binds a primate GM-CSF. In variousembodiments, the primate is selected from a monkey, a baboon, a macaque,a chimpanzee, a gorilla, a lemur, a lorise, a tarsier, a galago, apotto, a sifaka, an indri, an aye-ayes or an ape. In some embodiments,the anti-GM-CSF antibody binds a mammalian GM-CSF.

In particular embodiments, the anti-GM-CSF antibody is an anti-hGM-CSFantibody. As described above, the anti-GM-CSF antibody is a monoclonalantibody. In another embodiment, the anti-hGM-CSF antibody is anantibody fragment that is a Fab, a Fab′, a F(ab′)2, a scFv, or a dAB. Insome embodiments, the anti-hGM-CSF antibody is a human GM-CSFneutralizing antibody. In certain embodiments, the anti-hGM-CSF antibodyis a recombinant or chimeric antibody. In various embodiments, theanti-hGM-CSF antibody is a human antibody. In some embodiments, theanti-hGM-CSF antibody binds to the same epitope as chimeric 19/2antibody. In certain embodiments, the anti-hGM-CSF antibody comprisesthe VH region CDR3 and VL region CDR3 of chimeric 19/2 antibody. Invarious embodiments, the anti-hGM-CSF antibody comprises the VH regionand VL region CDR1, CDR2, and CDR3 of chimeric 19/2. In someembodiments, the anti-hGM-CSF antibody comprises a VH region thatcomprises a CDR3 binding specificity determinant RQRFPY (SEQ ID NO: 12)or RDRFPY (SEQ ID NO: 13), a J segment, and a V-segment, wherein theJ-segment comprises at least 95% identity to human JH4 (YFD YWGQGTLVTVSS (SEQ ID NO: 14)) and the V-segment comprises at least 90% identityto a human germ line VHl 1-02 or VHl 1-03 sequence; or a VH region thatcomprises a CDR3 binding specificity determinant RQRFPY(SEQ ID NO: 12).In particular embodiments, the J segment comprises YFDYWGQGTLVTVSS (SEQID NO: 14). In additional embodiments, the CDR3 comprises RQRFPYYFDY(SEQ ID NO: 15) or RDRFPYYFDY (SEQ ID NO: 16). In some embodiments, theVH region CDR1 is a human germline VHl CDR1; the VH region CDR2 is ahuman germline VHl CDR2; or both the CDR1 and CDR2 are from a humangermline VHl sequence.

In certain embodiments, the anti-hGM-CSF antibody comprises a VH CDR1,or a VH CDR2, or both a VH CDR1 and a VH CDR2 as shown in a VH regionset forth in FIG. 1. In various embodiments, the V-segment sequence hasa VH V segment sequence shown in FIG. 1. In certain embodiments, the VHhas the sequence of VH #1 (SEQ ID NO:1), VH #2 (SEQ ID NO:2), VH #3 (SEQID NO:3), VH #4 (SEQ ID NO:4), or VH #5 (SEQ ID NO:5) set forth inFIG. 1. In some embodiments, the anti-hGM-CSF antibody comprises aVL-region that comprises a CDR3 comprising the amino acid sequence FNKor FNR. In some embodiments, the anti-hGM-CSF antibody comprises a humangermline JK4 region. In certain embodiments, the VL region CDR3comprises QQFN(K/R)SPLT (SEQ ID NO: 17). In various embodiments, theanti-hGM-CSF antibody comprises a VL region that comprises a CDR3comprising QQFNKSPLT (SEQ ID NO: 18). In some embodiments, the VL regioncomprises a CDR1, or a CDR2, or both a CDR1 and CDR2 of a VL regionshown in FIG. 1. In particular embodiments, the VL region comprises a Vsegment that has at least 95% identity to the VKIII A27 (SEQ ID NO: 21)V-segment sequence as shown in FIG. 1. In some embodiments, the VLregion has the sequence of VK #1 (SEQ ID NO: 6), VK #2 (SEQ ID NO: 7),VK #3 (SEQ ID NO: 8), or VK #4 (SEQ ID NO: 9) set forth in FIG. 1. Incertain embodiments, the anti-hGM-CSF antibody has a VH region CDR3binding specificity determinant RQRFPY (SEQ ID NO: 12) or RDRFPY (SEQ IDNO: 13) and a VL region that has a CDR3 comprising QQFNKSPLT (SEQ ID NO:18). In some embodiments, the anti-hGM-CSF antibody has a VH regionsequence set forth in FIG. 1 and a VL region sequence set forth inFIG. 1. In other embodiments, the VH region or the VL region, or boththe VH and VL region amino acid sequences comprise a methionine at theN-terminus.

In some embodiments, the hGM-CSF antagonist is selected from the groupcomprising of an anti-hGM-CSF receptor antibody or a soluble hGM-CSFreceptor, a cytochrome b562 antibody mimetic, a hGM-CSF peptide analog,an adnectin, a lipocalin scaffold antibody mimetic, a calixareneantibody mimetic, and an antibody like binding peptidomimetic. Incertain embodiments, the CAR-T cells are CD19 CAR-T cells. In particularembodiments, the immunotherapy-related toxicity is CAR-T relatedtoxicity. In some embodiments, the CAR-T related toxicity is CRS, NT orneuro-inflammation.

In particular embodiments, the tumor relapse occurrence is reduced byfrom 50% to 100% in the first one-quarter of a year after administeringthe recombinant GM-CSF antagonist compared to tumor relapse occurrencein a subject treated with immunotherapy and not administered arecombinant GM-CSF antagonist. In certain embodiments, the tumor relapseoccurrence is reduced by from 50% to 95% in the first half-year afteradministering the recombinant GM-CSF antagonist. In various embodiments,the tumor relapse occurrence is reduced by from 50% to 90% in the firstyear after administering the recombinant GM-CSF antagonist. In someembodiments, the tumor relapse occurrence is prevented long-term. Asused herein the term “long-term” means during an extended period of timeof at least a year, i.e. 12 months, from the last date of treatment witha recombinant hGM-CSF antagonist. In some embodiments, the recombinanthGM-CSF antagonist is a hGM-CSF neutralizing antibody. In variousembodiments, the recombinant hGM-CSF antagonist is an anti-hGM-CSFantibody, e.g., Lenzilumab. In certain embodiments, the tumor relapseoccurrence is prevented by 12-36 months. In some embodiments, the tumorrelapse occurrence is prevented “completely” (100%), which as usedherein means that there is no recurrence of the tumor for at least 12months, from the last date of treatment with a recombinant hGM-CSFantagonist. In certain embodiments, the subject has acute lymphoblasticleukemia.

In various embodiments of the herein provided methods for reducingrelapse rate or preventing occurrence of tumor relapse in a subjecttreated with immunotherapy, the subject has a “refractory cancer”, whichas used herein is (a) a malignancy (also called “cancer” or a “tumor”herein) for which surgery is ineffective and is (b) either initiallyunresponsive or resistant to treatment, wherein the treatment ischemotherapy, radiation or a combination thereof, or is (b) a malignancywhich becomes or has become unresponsive to the aforementionedtreatment(s). In some embodiments, the subject has a “relapsed” cancer,which as used herein is a cancer that responded but to treatment, buthas returned. In particular embodiments, the refractory cancer or therelapsed cancer is non-Hodgkin lymphoma (NHL). In various embodiments,the refractory cancer or the relapsed cancer is non-Hodgkin lymphoma(NHL). In certain embodiments, the refractory cancer is refractoryaggressive B cell non-Hodgkin lymphoma. In some embodiments, therefractory cancer or the relapsed cancer is chemo-refractory B celllymphoma. In various embodiments, the refractory cancer or the relapsedcancer is hormone-refractory prostate cancer. In certain embodiments,the refractory cancer or the relapsed cancer is a pediatric cancer. Insome embodiments, the refractory pediatric cancer or the relapsedpediatric cancer is neuroblastoma. In particular embodiments, therefractory pediatric cancer or the relapsed pediatric cancer is apediatric leukemia selected from the group consisting of acutelymphoblastic leukemia (ALL), acute myelogenous leukemia (AML) or anuncommon pediatric leukemia which is juvenile myelomonocytic leukemia orchronic myeloid leukemia. In certain embodiments, the refractory canceror the relapsed cancer is a pediatric bone cancer. In some embodiments,the refractory cancer or the relapsed cancer is an adrenal cancer. Invarious embodiments, the refractory cancer or the relapsed cancer is abreast cancer. In certain embodiments, the refractory cancer or therelapsed cancer is a colon cancer, rectal cancer or colorectal cancer.In particular embodiments, the refractory cancer or the relapsed canceris a T-cell lymphoma. In some embodiments, the refractory cancer or therelapsed cancer is a head and neck cancer. In some embodiments, therefractory cancer or the relapsed cancer is a brain and/or spinal cordcancer, including but not limited to glioma an glioblastoma. Inadditional embodiments, the refractory cancer or the relapsed cancer isa tumor of bone or soft tissue, including but not limited to achondrosarcoma. In various embodiments, the refractory cancer or therelapsed cancer is a bone cancer. In some embodiments, the refractorycancer or the relapsed cancer is esophageal cancer. In certainembodiments, the refractory cancer or the relapsed cancer is a gallbladder cancer. In some embodiments, the refractory cancer or therelapsed cancer is a kidney cancer. In various embodiments, therefractory cancer or the relapsed cancer is melanoma. In someembodiments, the refractory cancer or the relapsed cancer is an ovarycancer. In certain embodiments, the refractory cancer or the relapsedcancer is a pancreatic cancer. In some embodiments, the refractorycancer or the relapsed cancer is a skin cancer selected from a basalcell carcinoma, a squamous cell carcinoma or a melanoma. In variousembodiments, the refractory cancer or the relapsed cancer is a lungcancer. In some embodiments, the refractory cancer or the relapsedcancer is a salivary gland cancer. In additional embodiments, therefractory cancer or the relapsed cancer is a uterine smooth musclecancer. In some embodiments, the refractory cancer or the relapsedcancer is a testicular cancer. In various embodiments, the refractorycancer or the relapsed cancer is a stomach cancer or a gastrointestinalcancer. In certain embodiments, the refractory cancer or the relapsedcancer is a bladder cancer. In additional embodiments, the refractorycancer or the relapsed cancer is an adipose tissue neoplasm. In someembodiments, the refractory pediatric cancer or the relapsed pediatriccancer is an adenocarcinoma. In certain embodiments, the refractorycancer or the relapsed cancer is a thymoma. In various embodiments, therefractory cancer or the relapsed cancer is an angiosarcoma, i.e., acancer of the lining of blood vessels, which can occur in any part ofthe body, including but not limited to skin, breast, liver, spleen anddeep tissue, i.e., deep-seated tumors. In some embodiments, therefractory cancer or the relapsed cancer is a metastasis of any one ofthe aforementioned refractory cancer or the relapsed cancer.

In some embodiments, the immunotherapy is an activation immunotherapy.In some embodiments, immunotherapy is provided as a cancer treatment. Insome embodiments, immunotherapy comprises adoptive cell transfer.

In some embodiments, adoptive cell transfer comprises administration ofa chimeric antigen receptor-expressing T-cell (CAR T-cell). A skilledartisan would appreciate that CARs are a type of antigen-targetedreceptor composed of intracellular T-cell signaling domains fused toextracellular tumor-binding moieties, most commonly single-chainvariable fragments (scFvs) from monoclonal antibodies. CARs directlyrecognize cell surface antigens, independent of MHC-mediatedpresentation, permitting the use of a single receptor construct specificfor any given antigen in all patients. Initial CARs fusedantigen-recognition domains to the CD3ζ activation chain of the T-cellreceptor (TCR) complex. While these first-generation CARs induced T-celleffector function in vitro, they were largely limited by poor antitumorefficacy in vivo. Subsequent CAR iterations have included secondarycostimulatory signals in tandem with CD3, including intracellulardomains from CD28 or a variety of TNF receptor family molecules such as4-1BB (CD137) and OX40 (CD134). Further, third generation receptorsinclude two costimulatory signals in addition to CD3ζ, most commonlyfrom CD28 and 4-1BB. Second and third generation CARs dramaticallyimprove antitumor efficacy, in some cases inducing complete remissionsin patients with advanced cancer. In one embodiment, a CAR T-cell is animmunoresponsive cell modified to express CARs, which is activated whenCARs bind to its antigen.

In one embodiment, a CAR T-cell is an immunoresponsive cell comprisingan antigen receptor, which is activated when its receptor binds to itsantigen. In one embodiment, the CAR T-cells used in the compositions andmethods as disclosed herein are first generation CAR T-cells. In anotherembodiment, the CAR T-cells used in the compositions and methods asdisclosed herein are second generation CAR T-cells. In anotherembodiment, the CAR T-cells used in the compositions and methods asdisclosed herein are third generation CAR T-cells. In anotherembodiment, the CAR T-cells used in the compositions and methods asdisclosed herein are fourth generation CAR T-cells.

In some embodiments, adoptive cell transfer comprises administeringT-cell receptor (TCR) modified T-cells. A skilled artisan wouldappreciate that TCR modified T-cells are manufactured by isolatingT-cells from tumor tissue and isolating their TCRα and TCRβ chains.These TCRα and TCRβ are later cloned and transfected into T cellsisolated from peripheral blood, which then express TCRα and TCRβ fromT-cells recognizing the tumor.

In some embodiments, adoptive cell transfer comprises administeringtumor infiltrating lymphocytes (TIL). In some embodiments, adoptive celltransfer comprises administering chimeric antigen receptor(CAR)-modified NK cells. A skilled artisan would appreciate thatCAR-modified NK cells comprise NK cells isolated from the patient orcommercially available NK engineered to express a CAR that recognizes atumor-specific protein.

In some embodiments, adoptive cell transfer comprises administeringdendritic cells.

In some embodiments, immunotherapy comprises administering monoclonalantibodies. In some embodiments, monoclonal antibodies attach tospecific proteins on cancer cells, thus flagging the cells for theimmune system finding and destroying them. In some embodiments,monoclonal antibodies work by inhibiting immune checkpoints, thushindering the inhibition of the immune system by cancer cells. In someembodiments, monoclonal antibodies improve utility of CAR-T to synergizewith checkpoint inhibitors.

In some embodiments, the antibody targets a protein selected from thegroup comprising: 5AC, 5T4, activin receptor-like kinase 1, AGS-22M6,alpha-fetoprotein, angiopoietin 2, angiopoietin 3, B7-H3, BAFF, BCMA,C242 antigen, CA-125, carbonic anhydrase 9, CCR4, CD125, CD152, CD184,CD19, CD2, CD20, CD200, CD22, CD221, CD23, CD25, CD27, CD274, CD276,CD28, CD3, CD30, CD33, CD37, CD38, CD4, CD40, CD41, CD44 v6, CD49b, CD5,CD51, CD52, CD54, CD56, CD6, CD70, CD74, CD79B, CD80, CEA, CFD, CGRP,ch4D5, CLDN18.2, clumping factor A, CSF1R, CSF2, CTGF, CTLA-4, DLL3,DLL4, DPP4, DR5, EGFL7, EGFR, endoglin, EpCAM, ephrin receptor A3,episialin, ERBB3 (HER3), FAP, FGF 23, fibrin II, beta chain, fibronectinextra domain-B, folate hydrolase, folate receptor, Frizzled receptor,GCGR, GD2 ganglioside, GD3 ganglioside, GDF-8, glypican 3, GM-CSF,GM-CSF receptor α-chain, GPNMB, GUCY2C, HER1, HER2/neu, HGF, HHGFR,histone complex, human scatter factor receptor kinase, human TNF, ICOSL,IFN-α, IGF1, IGF2, IGHE, IL-17A, IL-13, IL1A, IL-2, IL-6, IL-6 receptor,IL-8, IL-9, ILGF2, integrin α4, integrin α5β1, integrin α7 β7, integrinαvβ3, IP10, KIR2D, KLRC1, Lewis-Y antigen, MAGE-A, MCP-1, mesothelin,MIF, MIG, MIP1β, MS4A1, MSLN, MUC1, mucin CanAg, N-glycolylneuraminicacid, NOGO-A, Notch 1, Notch receptor, NRP1, OX-40, PD-1, PDCD1, PDGF-Rα, phosphate-sodium co-transporter, phosphatidylserine, platelet-derivedgrowth factor receptor beta, prostatic carcinoma cells, RHD, RON, RTN4,SDC1, sIL2Rα, SLAMF7, SOST, sphingosine-1-phosphate, Staphylococcusaureus, STEAP1, TAG-72, T-cell receptor, TEM1, tenascin C, TFPI, TGFbeta 1, TGF beta 2, TGF-β, TNFR superfamily member 4, TNF-α, TRAIL-R1,TRAIL-R2, TRP-1, TRP-2, TSLP, tumor antigen CTAA16.88, tumor specificglycosylation of MUC1, tumor-associated calcium signal transducer 2,TWEAK receptor, TYRP1 (glycoprotein 75), VEGFA, VEGFR-1, VEGFR2,vimentin, and VWF.

In some embodiments, the antibody is a bi-specific antibody. In someembodiments, the antibody is a bispecific T-cell engager (BiTE)antibody. In some embodiments, the antibody is selected from a groupcomprising: ipilimumab, nivolumab, pembrolizumab, atezolizumab,avelumab, durvalumab, rituximab, TGN1412, alemtuzumab, OKT3 or anycombination thereof.

In some embodiments, immunotherapy comprises administering cytokines. Askilled artisan would appreciate that cytokines can be administered inorder to enhance the immune system to attack the tumor by increasing itsrecognition and killing by immune cytotoxic cells. In some embodiments,the cytokine is selected from a group comprising: IFNα, IFNβ, IFNγ,IFNλ, IL-1, IL-2, IL-6, IL-7, IL-15, IL-21, IL-11, IL-12, IL-18, GM-CSF,TNFα, or any combination thereof.

In some embodiments, immunotherapy comprises administering immunecheckpoint inhibitors. A skilled artisan would appreciate that immunecheckpoints are membranal proteins that keep T cells from attacking thecells that express it. Immune checkpoints are often expressed by cancercells, thus preventing T cells from attacking them. In some embodiments,checkpoint proteins comprise PD-1/PD-L1 and CTLA-4/B7-1/B7-2. Blockingcheckpoint proteins was shown to disengage the inhibition of T cells toattack and kill cancer cells. In some embodiments, checkpoint inhibitorsare selected from a group comprising molecules blocking CTLA-4, PD-1, orPD-L1. In some embodiments, the checkpoint inhibitors are antibodies orparts thereof.

In some embodiments, immunotherapy comprises administeringpolysaccharides. A skilled artisan would appreciate that certainpolysaccharides found in mushroom enhance the immune system and itsanti-cancer properties. In some embodiments, polysaccharides arebeta-glucans or lentinan.

In some embodiments, immunotherapy comprises administering or a cancervaccine. A skilled artisan would appreciate that a cancer vaccineexposes the immune system to a cancer-specific antigen and an adjuvant.In some embodiments, the cancer vaccine is selected from a groupcomprising: sipuleucel-T, GVAX, ADXS11-001, ADXS31-001, ADXS31-164,ALVAC-CEA vaccine, AC Vaccine, talimogene laherparepvec, BiovaxID,Prostvac, CDX110, CDX1307, CDX1401, CimaVax-EGF, CV9104, DNDN, NeuVax,Ae-37, GRNVAC, tarmogens, GI-4000, GI-6207, GI-6301, ImPACT Therapy,IMA901, hepcortespenlisimut-L, Stimuvax, DCVax-L, DCVax-Direct, DCVaxProstate, CBLI, Cvac, RGSH4K, SCIB1, NCT01758328, and PVX-410.

Methods for Reducing a Level of a Cytokine or Chemokine Other thanGM-CSF

In some embodiments, inhibiting or reducing the incidence or theseverity of immunotherapy-related toxicity comprises decreasing theconcentration of at least one inflammation-associated factor in a bodyfluid. In some embodiments, inhibiting or reducing the incidence or theseverity of immunotherapy-related toxicity comprises decreasing theconcentration of at least one inflammation-associated factor in theserum. In some embodiments, inhibiting or reducing the incidence or theseverity of immunotherapy-related toxicity comprises decreasing theconcentration of at least one inflammation-associated factor in thecerebrospinal fluid (CSF). In some embodiments, disclosed herein aremethods for decreasing the concentration of at least oneinflammation-associated factor in serum. In some embodiments, disclosedherein are methods for decreasing the concentration of at least oneinflammation-associated factor in a tissue fluid. In some embodiments,disclosed herein are methods for decreasing the concentration of atleast one inflammation-associated factor in CSF. In some embodiments,the concentration of at least one inflammation-associated factor inserum is decreased. In some embodiments, the concentration of at leastone inflammation-associated factor in a tissue fluid is decreased. Insome embodiments, the concentration of at least oneinflammation-associated factor in CSF is decreased. A skilled artisanwould appreciate that decreasing the concentration of aninflammation-associated factor comprises decreasing or inhibiting theproduction of said inflammation-associated factor in a subject, orinhibiting or reducing the incidence or the severity ofimmunotherapy-related toxicity in a subject. In another embodiment,decreasing or inhibiting the production of an inflammation-associatedfactor comprises treating immunotherapy-related toxicity. In anotherembodiment, decreasing or inhibiting the production of aninflammation-associated factor comprises preventingimmunotherapy-related toxicity. In another embodiment, decreasing orinhibiting the production of an inflammation-associated factor levelscomprises alleviating immunotherapy-related toxicity. In anotherembodiment, decreasing or inhibiting the production of aninflammation-associated factor comprises amelioratingimmunotherapy-related toxicity.

In some embodiments, the inflammation-associated factor is a cytokine.In some embodiments, inhibiting or reducing the incidence or theseverity of immunotherapy-related toxicity comprises decreasing theconcentration of at least one cytokine in the serum. In someembodiments, inhibiting or reducing the incidence or the severity ofimmunotherapy-related toxicity comprises decreasing the concentration ofat least one cytokine in the CSF.

In some embodiments, the cytokine is hGM-CSF. In some embodiments, thecytokine is interleukin (IL)-1β. In some embodiments, the cytokine isIL-2. In some embodiments, the cytokine is sIL2Rα. In some embodiments,the cytokine is IL-5. In some embodiments, the cytokine is IL-6. In someembodiments, the cytokine is IL-8. In some embodiments, the cytokine isIL-10. In some embodiments, the cytokine is IP10. In some embodiments,the cytokine is IL-13. In some embodiments, the cytokine is IL-15. Insome embodiments, the cytokine is tumor necrosis factor α (TNFα). Insome embodiments, the cytokine is interferon γ (IFNγ). In someembodiments, the cytokine is monokine induced by gamma interferon (MIG).In some embodiments, the cytokine is macrophage inflammatory protein(MIP) 1β. In some embodiments, the cytokine is C-reactive protein. Insome embodiments, decreasing or inhibiting the production of cytokinelevels comprises decreasing or inhibiting the production of onecytokine. In some embodiments, decreasing or inhibiting the productionof cytokine levels comprises decreasing or inhibiting the production ofat least one cytokine. In some embodiments, decreasing or inhibiting theproduction of cytokine levels comprises decreasing or inhibiting theproduction of a number of cytokines.

In one aspect, this invention provides a method reducing a level of acytokine or chemokine other than GM-CSF in a subject having an incidenceof immunotherapy-related toxicity, the method comprising administeringto the subject a recombinant hGM-CSF antagonist, wherein the level ofthe cytokine or chemokine is reduced compared the level thereof in asubject administered an isotype control antibody during the incidence ofimmunotherapy-related toxicity. In some embodiments, the immunotherapycomprises adoptive cell transfer, administration of monoclonalantibodies, administration of a cancer vaccine, T cell engagingtherapies, or any combination thereof. In certain embodiments, theadoptive cell transfer comprises administering chimeric antigenreceptor-expressing T-cells (CAR T-cells), T-cell receptor (TCR)modified T-cells, tumor-infiltrating lymphocytes (TIL), chimeric antigenreceptor (CAR)-modified natural killer cells, or dendritic cells, or anycombination thereof. In some embodiments, the CAR-T cells are CD19 CAR-Tcells. In certain embodiments, the recombinant GM-CSF antagonist is anhGM-CSF antagonist. In various embodiments, the recombinant GM-CSFantagonist is an anti-GM-CSF antibody. In particular embodiments, theanti-GM-CSF antibody binds a human GM-CSF. In other embodiments, theanti-GM-CSF antibody binds a primate GM-CSF, as described above. In someembodiments, the anti-GM-CSF antibody binds a mammalian GM-CSF. Incertain embodiments, the anti-GM-CSF antibody is an anti-hGM-CSFantibody. In some embodiments, the anti-hGM-CSF antibody is a monoclonalantibody. In various embodiments, the anti-hGM-CSF antibody is anantibody fragment that is a Fab, a Fab′, a F(ab′)2, a scFv, or a dAB. Insome embodiments, the anti-hGM-CSF antibody is a human GM-CSFneutralizing antibody. In certain embodiments, the anti-hGM-CSF antibodyis a recombinant or chimeric antibody. In some embodiments, theanti-hGM-CSF antibody is a human antibody. In particular embodiments,the cytokine or chemokine is a human cytokine or chemokine selected fromthe group consisting of IP-10, IL-2, IL-3, IL-5, IL-1Ra, VEGF, TNF-a,FGF-2, IFN-γ, IL-12p40, IL-12p70, sCD40L, MDC, MCP-1, MIP-1a, MIP-1b ora combination thereof, as demonstrated in Example 22. In someembodiments, the cytokine or chemokine is selected from the groupconsisting of IL-1a, IL-1b, IL-2, IL-4, IL-6, IL-9, IL-10, IP-10, KC,MCP-1, MIP or a combination thereof (see Example 22). In certainembodiments, the subject has acute lymphoblastic leukemia.

In one embodiment, the methods disclosed herein do not affect theefficacy of the immunotherapy. In another embodiment, the methodsdisclosed herein reduce the efficacy of the immunotherapy by less thanabout 5%. In another embodiment, the methods disclosed herein reduce theefficacy of the immunotherapy by less than about 10%. In anotherembodiment, the methods disclosed herein reduce the efficacy of theimmunotherapy by less than about 15%. In another embodiment, the methodsdisclosed herein reduce the efficacy of the immunotherapy by less thanabout 20%. In another embodiment, the methods disclosed herein reducethe efficacy of the immunotherapy by less than about 50%.

In one embodiment, the methods described herein increase the efficacy ofthe immunotherapy. In one embodiment, increasing the efficacy allows forimprovement of the clinical management, patient outcomes, andtherapeutic index of the immunotherapy. In another embodiment, theincreased efficacy enables administration of higher immunotherapy doses.In another embodiment, the increased efficacy reduces hospitalizationstay and additional treatments and monitoring. In an embodiment, theimmunotherapy comprises CAR-T.

Any appropriate method of quantifying cytotoxicity can be used todetermine whether the immunotherapy efficacy remains substantiallyunchanged. For example, cytotoxicity can be quantified using a cellculture-based assay such as the cytotoxic assays described in theExamples. Cytotoxicity assays can employ dyes that preferentially stainthe DNA of dead cells. In other cases, fluorescent and luminescentassays that measure the relative number of live and dead cells in a cellpopulation can be used. For such assays, protease activities serve asmarkers for cell viability and cell toxicity, and a labeled cellpermeable peptide generates fluorescent signals that are proportional tothe number of viable cells in the sample. In another embodiment, ameasure of cytotoxicity may be qualitative. In another embodiment, ameasure of cytotoxicity may be quantitative.

In an embodiment, said increased efficacy comprises increased CAR-T cellexpansion, reduced number and/or activity of myeloid-derived suppressorcells (MDSC) that inhibit T-cell function, synergy with a checkpointinhibitor, or any combination thereof. In another embodiment, saidincreased CAR-T cell expansion comprises at least a 50% increasecompared to a control. In another embodiment, said increased CAR-T cellexpansion comprises at least a one quarter log expansion compared to acontrol. In another embodiment, said increased cell expansion comprisesat least a one-half log expansion compared to a control. In anotherembodiment, said increased cell expansion comprises at least a one logexpansion compared to a control. In another embodiment, said increasedcell expansion comprises a greater than one log expansion compared to acontrol.

In one embodiment, immunotherapy-related toxicity appears between 2 daysto 4 weeks after administration of immunotherapy. In one embodiment,immunotherapy-related toxicity appears between 0 to 2 days afteradministration of immunotherapy. In some embodiments, the hGM-CSFantagonist is administered to subjects at the same time as immunotherapyas prophylaxis. In another embodiment, the hGM-CSF antagonist isadministered to subjects 0-2 days after administration of immunotherapy.In another embodiment, the hGM-CSF antagonist is administered tosubjects 2-3 days after administration of immunotherapy. In anotherembodiment, the hGM-CSF antagonist is administered to subjects 7 daysafter administration of immunotherapy. In another embodiment, thehGM-CSF antagonist is administered to subjects 10 days afteradministration of immunotherapy. In another embodiment, the hGM-CSFantagonist is administered to subjects 14 days after administration ofimmunotherapy. In another embodiment, the hGM-CSF antagonist isadministered to subjects 2-14 days after administration ofimmunotherapy.

In another embodiment, the hGM-CSF antagonist is administered tosubjects 2-3 hours after administration of immunotherapy. In anotherembodiment, the hGM-CSF antagonist is administered to subjects 7 hoursafter administration of immunotherapy. In another embodiment, thehGM-CSF antagonist is administered to subjects 10 hours afteradministration of immunotherapy. In another embodiment, the GM-CSFantagonist is administered to subjects 14 hours after administration ofimmunotherapy. In another embodiment, the hGM-CSF antagonist isadministered to subjects 2-14 hours after administration ofimmunotherapy.

In an alternative embodiment, the hGM-CSF antagonist is administered tosubjects prior to immunotherapy as prophylaxis. In another embodiment,the hGM-CSF antagonist is administered to subjects 1 day beforeadministration of immunotherapy. In another embodiment, the hGM-CSFantagonist is administered to subjects 2-3 days before administration ofimmunotherapy. In another embodiment, the hGM-CSF antagonist isadministered to subjects 7 days before administration of immunotherapy.In another embodiment, the hGM-CSF antagonist is administered tosubjects 10 days before administration of immunotherapy. In anotherembodiment, the hGM-CSF antagonist is administered to subjects 14 daysbefore administration of immunotherapy. In another embodiment, thehGM-CSF antagonist is administered to subjects 2-14 days beforeadministration of immunotherapy.

In another embodiment, the hGM-CSF antagonist is administered tosubjects 2-3 hours before administration of immunotherapy. In anotherembodiment, the hGM-CSF antagonist is administered to subjects 7 hoursbefore administration of immunotherapy. In another embodiment, thehGM-CSF antagonist is administered to subjects 10 hours beforeadministration of immunotherapy. In another embodiment, the hGM-CSFantagonist is administered to subjects 14 hours before administration ofimmunotherapy. In another embodiment, the hGM-CSF antagonist isadministered to subjects 2-14 hours before administration ofimmunotherapy.

In another embodiment, the hGM-CSF antagonist may be administeredtherapeutically, once immunotherapy-related toxicity has occurred. Inone embodiment, the hGM-CSF antagonist may be administered oncepathophysiological processes leading up to or attesting to the beginningof immunotherapy-related toxicity are detected. In one embodiment, thehGM-CSF antagonist can terminate the pathophysiological processes andavoid its sequelae. In some embodiments, the pathophysiologicalprocesses comprise at least one of the following: increased cytokineconcentrations in serum, increased cytokine concentrations in CSF,increased C-reactive protein (CRP) in serum, increased ferritin in theserum, increased IL-6 in serum, endothelial activation, disseminatedintravascular coagulation (DIC), increased ANG2 serum concentration,increased ANG2:ANG1 ratio in serum, CAR T-cell presence in CSF,increased Von Willebrand factor (VWF) serum concentration,blood-brain-barrier (BBB) leakage, or any combination thereof.

In another embodiment, the hGM-CSF antagonist may be administeredtherapeutically, at multiple time points. In another embodiment,administration of the hGM-CSF antagonist is at least at two time points.In another embodiment, administration of the hGM-CSF antagonist is atleast at three time points.

In one embodiment, the hGM-CSF antagonist is administered once. Inanother embodiment, the hGM-CSF antagonist is administered twice. Inanother embodiment, the hGM-CSF antagonist is administered three times.In another embodiment, the hGM-CSF antagonist is administered fourtimes. In another embodiment, the hGM-CSF antagonist is administered atleast four times. In another embodiment, the hGM-CSF antagonist isadministered more than four times.

A skilled artisan would appreciate that immunotherapy-related toxicityis managed by different treatments. In some embodiments, the hGM-CSFantagonist is co-administered with other treatments. In someembodiments, other treatments are selected from a group comprising:cytokine-directed therapy, anti-IL-6 therapy, corticosteroids,tocilizumab, siltuximab, low-dose vasopressors, inotropic agents,supplemental oxygen, diuresis, thoracentesis, antiepileptics,benzodiazepines, levetiracetam, phenobarbital, hyperventilation,hyperosmolar therapy, and standard therapies for specific organtoxicities.

In some embodiments, immunotherapy-related toxicity comprises a braindisease, damage or malfunction. In some embodiments,immunotherapy-related toxicity comprises CAR T-cell related NT. In someembodiments, immunotherapy-related toxicity comprises CAR T-cell-relatedencephalopathy syndrome (CRES). In some embodiments, provided hereinmethods for inhibiting or reducing the incidence of a brain disease,damage or malfunction.

In some embodiments, inhibiting or reducing the incidence of CREScomprises ameliorating headaches. In some embodiments, inhibiting orreducing the incidence of CRES comprises alleviating delirium. In someembodiments, inhibiting or reducing the incidence of CRES comprisesreducing anxiety. In some embodiments, inhibiting or reducing theincidence of CRES comprises reducing tremors. In some embodiments,inhibiting or reducing the incidence of CRES comprises decreasingseizure activity. In some embodiments, inhibiting or reducing theincidence of CRES comprises decreasing confusion. In some embodiments,inhibiting or reducing the incidence of CRES comprises reducingalterations in wakefulness.

In some embodiments, inhibiting or reducing the incidence of CREScomprises reducing hallucinations. In some embodiments, inhibiting orreducing the incidence of CRES comprises reducing dysphasia. In someembodiments, inhibiting or reducing the incidence of CRES comprisesreducing ataxia. In some embodiments, inhibiting or reducing theincidence of CRES comprises reducing apraxia. In some embodiments,inhibiting or reducing the incidence of CRES comprises amelioratingfacial nerve palsy. In some embodiments, inhibiting or reducing theincidence of CRES comprises reducing motor weakness. In someembodiments, inhibiting or reducing the incidence of CRES comprisesreducing seizures. In some embodiments, inhibiting or reducing theincidence of CRES comprises reducing non-convulsive EEG seizures. Insome embodiments, inhibiting or reducing the incidence or severity ofCRES comprises improving coma recovery.

In some embodiments, inhibiting or reducing the incidence or severity ofCRES comprises reducing endothelial activation. A skilled artisan wouldappreciate that endothelial activation is an inflammatory andprocoagulant state of endothelial cells characterized by increasedinteractions with leukocytes.

In some embodiments, inhibiting or reducing the incidence of CREScomprises reducing vascular leak. The term “vascular leak” may be usedinterchangeably with the terms “vascular leak syndrome” and “capillaryleak syndrome” having all the same qualities and meanings. A skilledartisan would appreciate that vascular leak is associated withendothelial cells are separated allowing a leakage of plasma andtransendothelial migration of inflammatory cells into body tissues,resulting in tissue and organ damage. In addition, neutrophils can causemicrocirculatory occlusion, leading to decreased tissue perfusion. Insome embodiments reducing the incidence of CRES comprises reducingintravascular coagulation.

In some embodiments, inhibiting or reducing the incidence of CREScomprises reducing the concentration of at least one circulatingcytokine. In some embodiments, the cytokine is selected from a groupcomprising: hGM-CSF, IFNγ, IL-1, IL-15, IL-6, IL-8, IL-10, and IL-2. Insome embodiments, inhibiting or reducing the incidence of CRES comprisesreducing serum concentration of ANG2. In some embodiments, inhibiting orreducing the incidence of CRES comprises reducing ANG2:ANG1 ratio inserum.

In some embodiments, inhibiting or reducing the incidence of CREScomprises reducing the CRES grade. In some embodiments, inhibiting orreducing the incidence of CRES comprises improving CARTOX-10 score. Insome embodiments, inhibiting or reducing the incidence of CRES comprisesreducing a raise in intracranial pressure. In some embodiments,inhibiting or reducing the incidence of CRES comprises reducingseizures. In some embodiments, inhibiting or reducing the incidence ofCRES comprises reducing motor weakness.

In some embodiments, immunotherapy-related toxicity comprises CAR T-cellrelated CRS. In some embodiments, provided herein are methods forinhibiting or reducing the incidence or severity of CRS and/or NT.

In some embodiments, inhibiting or reducing the incidence of CRS or NTcomprises, without limitation, ameliorating fever (with or withoutrigors, malaise, fatigue, anorexia, myalgia, arthralgia, nausea,vomiting, headache, skin rash, diarrhea, tachypnea, hypoxemia, hypoxia,shock, cardiovascular tachycardia, widened pulse pressure, hypotension,capillary leak, increased early cardiac output, diminished late cardiacoutput, elevated D-dimer, hypofibrinogenemia with or without bleeding,azotemia, transaminitis, hyperbilirubinemia, mental status changes,confusion, delirium, frank aphasia, hallucinations, tremor, dysmetria,altered gait, seizures, organ failure, or any combination thereof, orany other symptom or characteristic known in the art to be associatedwith CRS.

In some embodiments, inhibiting or reducing the incidence of CRScomprises reducing the concentration of at least one circulatingcytokine. In some embodiments, the cytokine is selected from a groupcomprising: GM-CSF, IFNγ, IL-1, IL-15, IL-6, IL-8, IL-10, and IL-2.

In some embodiments, inhibiting or reducing the incidence of CRScomprises reducing the CRS grade. In some embodiments, inhibiting orreducing the incidence of NT comprises reducing the NT grade. In someembodiments, inhibiting or reducing the incidence of CRS comprisesimproving CARTOX-10 score. In some embodiments, inhibiting or reducingthe incidence of NT comprises improving CARTOX-10 score. In someembodiments, inhibiting or reducing the incidence of CRS comprisesreducing raised intracranial pressure. In some embodiments, inhibitingor reducing the incidence of CRS comprises reducing seizures. In someembodiments, inhibiting or reducing the incidence of CRS comprisesreducing motor weakness. In some embodiments, inhibiting or reducing theincidence of NT or CRS comprises inhibiting or reducing the incidence toless than 60%. In some embodiments, inhibiting or reducing the incidenceof NT or CRS comprises inhibiting or reducing the incidence to less than50%. In some embodiments, inhibiting or reducing the incidence of NT orCRS comprises inhibiting or reducing the incidence to less than 40%. Insome embodiments, inhibiting or reducing the incidence of NT or CRScomprises inhibiting or reducing the incidence to less than 30%. In someembodiments, inhibiting or reducing the incidence of NT or CRS comprisesinhibiting or reducing the incidence to less than 20% of patients. Insome embodiments, inhibiting or reducing the incidence of NT or CRScomprises eliminating NT or CRS.

In some embodiments, the subject has Grade 1 CRS and/or NT. In someembodiments, the subject has Grade 2 CRS and or NT. In some embodiments,the subject has Grade 3 CRS and/or NT. In some embodiments, the subjecthas Grade 4 CRS and/or NT. In some embodiments, the subject has anycombination of the above.

In some embodiments, inhibiting or reducing the incidence of NT or CRScomprises reducing the CRS grade, the NT grade, or both. In someembodiments, the grade is reduced to ≤3 NT and/or CRS in 95% ofpatients.

In some embodiments, the subject has a body temperature above 37° C.following immunotherapy administration. In some embodiments, the subjecthas a body temperature above 38° C. following immunotherapyadministration. In some embodiments, the subject has a body temperatureabove 39° C. following immunotherapy administration. In someembodiments, the subject has a body temperature above 40° C. followingimmunotherapy administration. In some embodiments, the subject has abody temperature above 41° C. following immunotherapy administration. Insome embodiments, the subject has a body temperature above 42° C.following immunotherapy administration.

In some embodiments, the subject has IL-6 serum concentration above 10pg/mL following immunotherapy administration. In some embodiments, thesubject has IL-6 serum concentration above 12 pg/mL followingimmunotherapy administration. In some embodiments, the subject has IL-6serum concentration above 14 pg/mL following immunotherapyadministration. In some embodiments, the subject has IL-6 serumconcentration above 16 pg/mL following immunotherapy administration. Insome embodiments, the subject has IL-6 serum concentration above 18pg/mL following immunotherapy administration. In some embodiments, thesubject has IL-6 serum concentration above 20 pg/mL followingimmunotherapy administration. In some embodiments, the subject has IL-6serum concentration above 22 pg/mL following immunotherapyadministration.

In some embodiments, the subject has an MCP-1 serum concentration above200 pg/ml following immunotherapy administration. In some embodiments,the subject has an MCP-1 serum concentration above 400 pg/ml followingimmunotherapy administration. In some embodiments, the subject has anMCP-1 serum concentration above 600 pg/ml following immunotherapyadministration. In some embodiments, the subject has an MCP-1 serumconcentration above 800 pg/ml following immunotherapy administration. Insome embodiments, the subject has an MCP-1 serum concentration above1000 pg/ml following immunotherapy administration. In some embodiments,the subject has an MCP-1 serum concentration above 1200 pg/ml followingimmunotherapy administration. In some embodiments, the subject has anMCP-1 serum concentration above 1400 pg/ml following immunotherapyadministration. In some embodiments, the subject has an MCP-1 serumconcentration above 1600 pg/ml following immunotherapy administration.In some embodiments, the subject has an MCP-1 serum concentration above1800 pg/ml following immunotherapy administration. In some embodiments,the subject has an MCP-1 serum concentration above 2000 pg/ml followingimmunotherapy administration.

In some embodiments, the subject has Grade 1 CRES. In some embodiments,the subject has Grade 2 CRES. In some embodiments, the subject has Grade3 CRES. In some embodiments, the subject has Grade 4 CRES.

In some embodiments, the subject is predisposed to have a brain disease,damage or malfunction prior to immunotherapy. In some embodiments, thepredisposition is genetic. In some embodiments, the predisposition isacquired. In some embodiments, the predisposition regards an existingmedical condition. In some embodiments, the predisposition is diagnosedprior to immunotherapy. In some embodiments, the predisposition is notdiagnosed. In some embodiments, the subject goes through medicalevaluations in order to determine predisposition to acquire animmunotherapy-related brain disease, damage or malfunction prior toimmunotherapy.

In some embodiments, medical evaluations comprise determining ANG1concentration in a body fluid. In some embodiments, medical evaluationscomprise determining ANG1 concentration in serum. In some embodiments,medical evaluations comprise determining ANG2 concentration in a bodyfluid. In some embodiments, medical evaluations comprise determiningANG2 concentration in serum. In some embodiments, medical evaluationscomprise calculating the ANG2:ANG1 ratio in serum. In some embodiments,subjects with serum ANG2:ANG1 ratio above 0.5 prior to immunotherapy arepredisposed to CRES. In some embodiments, subjects with serum ANG2:ANG1ratio above 0.7 prior to immunotherapy are predisposed to CRES. In someembodiments, subjects with serum ANG2:ANG1 ratio above 0.9 prior toimmunotherapy are predisposed to CRES. In some embodiments, subjectswith serum ANG2:ANG1 ratio above 1 prior to immunotherapy arepredisposed to CRES. In some embodiments, subjects with serum ANG2:ANG1ratio above 1.1 prior to immunotherapy are predisposed to CRES. In someembodiments, subjects with serum ANG2:ANG1 ratio above 1.3 prior toimmunotherapy are predisposed to CRES. In some embodiments, subjectswith serum ANG2:ANG1 ratio above 1.5 prior to immunotherapy arepredisposed to CRES.

In some embodiments, immunotherapy-related toxicity compriseshemophagocytic lymphohistiocytosis (HLH). In some embodiments,immunotherapy-related toxicity comprises macrophage-activation syndrome(MAS). In some embodiments, provided herein methods for inhibiting orreducing the incidence of HLH. In some embodiments, provided hereinmethods for inhibiting or reducing the incidence of MAS.

In some embodiments, inhibiting or reducing the incidence of HLHcomprises increasing survival of the subject. In some embodiments,inhibiting reducing the incidence of HLH comprises increasing time torelapse. In some embodiments, inhibiting or reducing the incidence ofMAS comprises increasing survival of the subject. In some embodiments,inhibiting reducing the incidence of MAS comprises increasing time torelapse.

In some embodiments, inhibiting or reducing the incidence of HLH or MAScomprises inhibiting macrophage activation and/or proliferation. In someembodiments, inhibiting or reducing the incidence of HLH or MAScomprises inhibiting T lymphocytes activation and/or proliferation. Insome embodiments, inhibiting or reducing the incidence of HLH or MAScomprises reducing the concentration of circulating IFNγ. In someembodiments, inhibiting or reducing the incidence of HLH or MAScomprises reducing the concentration of circulating of GM-CSF.

In some embodiments the subject presents with fever followingimmunotherapy. In some embodiments the subject presents withsplenomegaly following immunotherapy. In some embodiments the subjectpresents with cytopenia following immunotherapy. In some embodiments thesubject presents with cytopenia in two or more cell lines followingimmunotherapy. In some embodiments the subject presents withhypertriglyceridemia following immunotherapy. In some embodiments thesubject presents with hypofibrinogenemia following immunotherapy. Insome embodiments the subject presents with hemophagocytosis followingimmunotherapy. In some embodiments hemophagocytosis is observed in bonemarrow. In some embodiments the subject presents with low NK-cellactivity following immunotherapy. In some embodiments the subjectpresents with absent NK activity following immunotherapy.

In some embodiments the subject presents with ferritin serumconcentrations above 100 U/ml following immunotherapy. In someembodiments the subject presents with ferritin serum concentrationsabove 300 U/ml following immunotherapy. In some embodiments the subjectpresents with ferritin serum concentrations above 500 U/ml followingimmunotherapy. In some embodiments the subject presents with ferritinserum concentrations above 700 U/ml following immunotherapy. In someembodiments the subject presents with ferritin serum concentrationsabove 900 U/ml following immunotherapy.

In some embodiments the subject presents with soluble CD25 serumconcentration above 1200 U/ml following immunotherapy. In someembodiments the subject presents with soluble CD25 serum concentrationabove 1500 U/ml following immunotherapy. In some embodiments the subjectpresents with soluble CD25 serum concentration above 1800 U/ml followingimmunotherapy. In some embodiments the subject presents with solubleCD25 serum concentration above 2000 U/ml following immunotherapy. Insome embodiments the subject presents with soluble CD25 serumconcentration above 2200 U/ml following immunotherapy. In someembodiments the subject presents with soluble CD25 serum concentrationabove 2400 U/ml following immunotherapy. In some embodiments the subjectpresents with soluble CD25 serum concentration above 2700 U/ml followingimmunotherapy. In some embodiments the subject presents with solubleCD25 serum concentration above 3000 U/ml following immunotherapy.

In some embodiments, the subject is predisposed to have HLH. In someembodiments, the predisposition is genetic. In some embodiments, thepredisposition regards an existing medical condition. A skilled artisanwould appreciate that sporadic HLH has been associated with a number ofgenetic mutations. In some embodiments, the subject carries a mutationin a gene selected from PRF1, UNC13D, STX11, STXBP2, or RAB27A, or anycombination thereof. In some embodiments, the subject has reduced orabsent expression of perforin.

hGM-CSF Antagonists

hGM-CSF antagonists suitable for use selectively interfere with theinduction of signaling by the hGM-CSF receptor by causing a reduction inthe binding of hGM-CSF to the receptor. Such antagonists may includeantibodies that bind the hGM-CSF receptor, antibodies that bind tohGM-CSF, GM-CSF analogs such as E21R, and other proteins or smallmolecules that compete for binding of hGM-CSF to its receptor or inhibitsignaling that normally results from the binding of the ligand to thereceptor.

In many embodiments, the hGM-CSF antagonist used in the invention is apolypeptide e.g., an anti-hGM-CSF antibody, an anti-hGM-CSF receptorantibody, a soluble hGM-CSF receptor, or a modified GM-CSF polypeptidethat competes for binding with hGM-CSF to a receptor, but is inactive.Such proteins are often produced using recombinant expressiontechnology. Such methods are widely known in the art. General molecularbiology methods, including expression methods, can be found, e.g., ininstruction manuals, such as, Sambrook and Russell (2001) MolecularCloning: A laboratory manual 3rd ed. Cold Spring Harbor LaboratoryPress; Current Protocols in Molecular Biology (2006) John Wiley and SonsISBN: 0-471-50338-X.

A variety of prokaryotic and/or eukaryotic based protein expressionsystems may be employed to produce a hGM-CSF antagonist protein. Manysuch systems are widely available from commercial suppliers.

hGM-CSF Antibodies

The hGM-CSF antibodies of the present invention are antibodies that bindwith high affinity to hGM-CSF and are antagonists of hGM-CSF. Theantibodies comprise variable regions with a high degree of identity tohuman germ-line V_(H) and V_(L) sequences. In preferred embodiments, theBSD sequence in CDRH3 of an antibody of the invention comprises theamino acid sequence RQRFPY (SEQ ID NO: 12) or RDRFPY(SEQ ID NO: 13). TheBSD in CDRL3 comprises FNK or FNR.

Complete V-regions are generated in which the BSD forms part of the CDR3and additional sequences are used to complete the CDR3 and add a FR4sequence. Typically, the portion of the CDR3 excluding the BSD and thecomplete FR4 are comprised of human germ-line sequences. In someembodiments, the CDR3-FR4 sequence excluding the BSD differs from humangerm-line sequences by not more than 2 amino acids on each chain. Insome embodiments, the J-segment comprises a human germline J-segment.Human germline sequences can be determined, for example, through thepublicly available international ImMunoGeneTics database (IMGT) andV-base (on the worldwide web at vbase.mrc-cpe.cam.ac.uk).

The human germline V-segment repertoire consists of 51 heavy chainV-regions, 40 K light chain V-segments, and 31λ light chain V-segments,making a total of 3,621 germline V-region pairs, in addition, there arestable allelic variants for most of these V-segments, but thecontribution of these variants to the structural diversity of thegermline repertoire is limited. The sequences of all human germ-lineV-segment genes are known and can be accessed in the V-base database,provided by the MRC Centre for Protein Engineering, Cambridge, UnitedKingdom (see, also Chothia et al., 1992, J Mol Biol 227:776-798;Tomlinson et al., 1995, EMBO J 14:4628-4638; and Williams et al., 1996,J Mol Biol 264:220-232).

Antibodies or antibodies fragments as described herein can be expressedin prokaryotic or eukaryotic microbial systems or in the cells of highereukaryotes such as mammalian cells.

An antibody that is employed in the invention can be in any format. Forexample, in some embodiments, the antibody can be a complete antibodyincluding a constant region, e.g., a human constant region, or can be afragment or derivative of a complete antibody, e.g., an Fd, a Fab, Fab′,F(ab′)₂, scFv, Fv, an Fv fragment, or a single domain antibody, such asa nanobody or a camelid antibody. Such antibodies may additionally berecombinantly engineered by methods well known to persons of skill inthe art. As noted above, such antibodies can be produced using knowntechniques.

In some embodiments, the hGM-CSF antagonist is an antibody that binds tohGM-CSF or an antibody that binds to the hGM-CSF receptor α or βsubunit. The antibodies can be raised against hGM-CSF (or hGM-CSFreceptor) proteins, or fragments, or produced recombinantly. Antibodiesto GM-CSF for use in the invention can be neutralizing or can benon-neutralizing antibodies that bind GM-CSF and increase the rate of invivo clearance of hGM-CSF such that the hGM-CSF level in the circulationis reduced. Often, the hGM-CSF antibody is a neutralizing antibody.

Methods of preparing polyclonal antibodies are known to the skilledartisan (e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988);Methods in Immunology). Polyclonal antibodies can be raised in a mammalby one or more injections of an immunizing agent and, if desired, anadjuvant. The immunizing agent includes a GM-CSF or GM-CSF receptorprotein, e.g., a human GM-CSF or GM-CSF receptor protein, or fragmentthereof.

In some embodiment, a GM-CSF antibody for use in the invention ispurified from human plasma. In such embodiments, the GM-CSF antibody istypically a polyclonal antibody that is isolated from other antibodiespresent in human plasma. Such an isolation procedure can be performed,e.g., using known techniques, such as affinity chromatography.

In some embodiments, the GM-CSF antagonist is a monoclonal antibody.

Monoclonal antibodies may be prepared using hybridoma methods, such asthose described by Kohler & Milstein, Nature 256:495 (1975). In ahybridoma method, a mouse, hamster, or other appropriate host animal, istypically immunized with an immunizing agent, such as human GM-CSF, toelicit lymphocytes that produce or are capable of producing antibodiesthat will specifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro. The immunizing agent preferablyincludes human GM-CSF protein, fragments thereof, or fusion proteinthereof.

Human monoclonal antibodies can be produced using various techniquesknown in the art, including phage display libraries (Hoogenboom &Winter, J. Mol. Biol. 227:381 (1991); Marks et al, J. Mol. Biol. 222:581(1991)). The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, p. 77 (1985) and Boerneret al., J. Immunol. 147(1):86-95 (1991)). Similarly, human antibodiescan be made by introducing of human immunoglobulin loci into transgenicanimals, e.g., mice in which the endogenous immunoglobulin genes havebeen partially or completely inactivated. Upon challenge, human antibodyproduction is observed, which closely resembles that seen in humans inall respects, including gene rearrangement, assembly, and antibodyrepertoire. This approach is described, e.g., in U.S. Pat. Nos.5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and inthe following scientific publications: Marks et al., Bio/Technology10:779-783 (1992); Lonberg et al, Nature 368:856-859 (1994); Morrison,Nature 368:812-13 (1994); Fishwild et al, Nature Biotechnology 14:845-51(1996); Neuberger, Nature Biotechnology 14:826 (1996); Lonberg & Huszar,Intern. Rev. Immunol. 13:65-93 (1995).

In some embodiments the anti-GM-CSF antibodies are chimeric or humanizedmonoclonal antibodies. As noted supra, humanized forms of antibodies arechimeric immunoglobulins in which residues from a complementarydetermining region (CDR) of human antibody are replaced by residues froma CDR of a non-human species such as mouse, rat or rabbit having thedesired specificity, affinity and capacity.

In some embodiments of the invention, the antibody is additionallyengineered to reduced immunogenicity, e.g., so that the antibody issuitable for repeat administration. Methods for generating antibodieswith reduced immunogenicity include humanization/humaneering proceduresand modification techniques such as de-immunization, in which anantibody is further engineered, e.g., in one or more framework regions,to remove T cell epitopes.

In some embodiments, the antibody is a humaneered antibody. A humaneeredantibody is an engineered human antibody having a binding specificity ofa reference antibody, obtained by joining a DNA sequence encoding abinding specificity determinant (BSD) from the CDR3 region of the heavychain of the reference antibody to human VH segment sequence and a lightchain CDR3 BSD from the reference antibody to a human VL segmentsequence. Methods for Humaneering are provided in US patent applicationpublication no. 20050255552 and US patent application publication no.20060134098. Methods for signal-less secretion of antibody fragmentsfrom E. coli are described in US patent application 20070020685.

An antibody can further be de-immunized to remove one or more predictedT-cell epitopes from the V-region of an antibody. Such procedures aredescribed, for example, in WO 00/34317.

The heavy chain constant region is often a gamma chain constant region,for example, a gamma-1, gamma-2, gamma-3, or gamma-4 constant region. Insome embodiments, e.g., where the antibody is a fragment, the antibodycan be conjugated to another molecule, e.g., to provide an extendedhalf-life in vivo such as a polyethylene glycol (pegylation) or serumalbumin. Examples of PEGylation of antibody fragments are provided inKnight et al (2004) Platelets 15: 409 (for abciximab); Pedley et al(1994) Br. J. Cancer 70: 1126 (for an anti-CEA antibody) Chapman et al(1999) Nature Biotech. 17: 780.

An antibody for use in the invention binds to hGM-CSF or hGM-CSFreceptor. Any number of techniques can be used to determine antibodybinding specificity. See, e.g., Harlow & Lane, Antibodies, A LaboratoryManual (1988) for a description of immunoassay formats and conditionsthat can be used to determine specific immunoreactivity of an antibody.

An exemplary antibody suitable for use with the present invention iscl9/2 (a mouse/69 human chimeric anti-hGM-CSF antibody). In someembodiments, a monoclonal antibody that competes for binding to the sameepitope as cl9/2, or that binds the same epitope as cl9/2, is used. Theability of a particular antibody to recognize the same epitope asanother antibody is typically determined by the ability of the firstantibody to competitively inhibit binding of the second antibody to theantigen. Any of a number of competitive binding assays can be used tomeasure competition between two antibodies to the same antigen. Forexample, a sandwich ELISA assay can be used for this purpose. This iscarried out by using a capture antibody to coat the surface of a well. Asubsaturating concentration of tagged-antigen is then added to thecapture surface. This protein will be bound to the antibody through aspecific antibody-epitope interaction. After washing a second antibody,which has been covalently linked to a detectable moiety (e.g., HRP, withthe labeled antibody being defined as the detection antibody) is addedto the ELISA. If this antibody recognizes the same epitope as thecapture antibody it will be unable to bind to the target protein as thatparticular epitope will no longer be available for binding. If, howeverthis second antibody recognizes a different epitope on the targetprotein it will be able to bind and this binding can be detected byquantifying the level of activity (and hence antibody bound) using arelevant substrate. The background is defined by using a single antibodyas both capture and detection antibody, whereas the maximal signal canbe established by capturing with an antigen specific antibody anddetecting with an antibody to the tag on the antigen. By using thebackground and maximal signals as references, antibodies can be assessedin a pair-wise manner to determine epitope specificity.

A first antibody is considered to competitively inhibit binding of asecond antibody, if binding of the second antibody to the antigen isreduced by at least 30%, usually at least about 40%, 50%, 60% or 75%,and often by at least about 90%, in the presence of the first antibodyusing any of the assays described above.

In some embodiments of the invention, an antibody is employed thatcompetes with binding, or bind, to the same epitope as a known antibody,e.g., cl9/2. Method of mapping epitopes are well known in the art. Forexample, one approach to the localization of functionally active regionsof human granulocyte-macrophage colony-stimulating factor (hGM-CSF) isto map the epitopes recognized by neutralizing anti-hGM-CSF monoclonalantibodies. For example, the epitope to which cl9/2 (which has the samevariable regions as the neutralizing antibody LMM 102) binds has beendefined using proteolytic fragments obtained by enzymic digestion ofbacterially synthesized hGM-CSF (Dempsey, et al, Hybridoma 9:545-558,1990). RP-HPLC fractionation of a tryptic digest resulted in theidentification of an immunoreactive “tryptic core” peptide containing 66amino acids (52% of the protein). Further digestion of this “trypticcore” with S. aureus V8 protease produced a unique immunoreactivehGM-CSF product comprising two peptides, residues 86-93 and 112-127,linked by a disulfide bond between residues 88 and 121. The individualpeptides were not recognized by the antibody.

In some embodiments, the antibodies suitable for use with the presentinvention have a high affinity binding for human GM-CSF or hGM-CSFreceptor. High affinity binding between an antibody and an antigenexists if the dissociation constant (KD) of the antibody is <about 10nM, typically <1 nM, and preferably <100 pM. In some embodiments, theantibody has a dissociation rate of about 10⁻⁴ per second or better.

A variety of methods can be used to determine the binding affinity of anantibody for its target antigen such as surface plasmon resonanceassays, saturation assays, or immunoassays such as ELISA or RIA, as arewell known to persons of skill in the art. An exemplary method fordetermining binding affinity is by surface plasmon resonance analysis ona BIAcore™ 2000 instrument (Biacore AB, Freiburg, Germany) using CM5sensor chips, as described by Krinner et al, (2007) Mol. Immunol.February; 44(5):916-25. (Epub 2006 May H)).

In some embodiments, the hGM-CSF antagonists are neutralizing antibodiesto hGM-CSF, its receptor or its receptor subunit, which bind in a mannerthat interferes with the binding of hGM-CSF to its receptor or receptorsubunit. In some embodiments, an anti-hGM-CSF antibody for use in theinvention inhibits binding to the alpha subunit of the hGM-CSF receptor.Such an antibody can, for example, bind to hGM-CSF at the region wherehGM-CSF binds to the receptor and thereby inhibit binding. In anotherembodiments, the anti-hGM-CSF antibody inhibits hGM-CSF functioningwithout blocking its binding to the alpha subunit of the hGM-CSFreceptor.

II. Heavy Chains

A heavy chain of an anti-hGM-CSF antibody of the invention comprises aheavy-chain V-region that comprises the following elements:

1) human heavy-chain V-segment sequences comprisingFR1-CDR1-FR2-CDR2-FR3

2) a CDRH3 region comprising the amino acid sequence R(Q/D)RFPY (SEQ IDNO: 22)

3) a FR4 contributed by a human germ-line J-gene segment.

Examples of V-segment sequences that support binding to hGM-CSF incombination with a CDR3-FR4 segment described above together with acomplementary VL region are shown in FIG. 1. The V-segments can be,e.g., from the human VH1 subclass. In some embodiments, the V-segment isa human V_(H)1 sub-class segment that has a high degree of amino-acidsequence identity, e.g., at least 80%, 85%, or 90% or greater identity,to the germ-line segment VH1 1-02 (SEQ ID NO: 19) or VH1 1-03 (SEQ IDNO: 20). In some embodiments, the V-segment differs by not more than 15residues from VH1 1-02 (SEQ ID NO: 19) or VH1 1-03 (SEQ ID NO: 20) andpreferably not more than 7 residues.

The FR4 sequence of the antibodies of the invention is provided by ahuman JH1, JH3, JH4, JH5 or JH6 gene germline segment, or a sequencethat has a high degree of amino-acid sequence identity to a humangermline JH segment. In some embodiments, the J segment is a humangermline JH4 sequence.

The CDRH3 also comprises sequences that are derived from a humanJ-segment. Typically, the CDRH3-FR4 sequence excluding the BSD differsby not more than 2 amino acids from a human germ-line J-segment. Intypical embodiments, the J-segment sequences in CDRH3 are from the sameJ-segment used for the FR4 sequences. Thus, in some embodiments, theCDRH3-FR4 region comprises the BSD and a complete human JH4 germ-linegene segment. An exemplary combination of CDRH3 and FR4 sequences isshown below, in which the BSD is in bold and human germ-line J-segmentJH4 residues are underlined:

CDR3 (SEQ ID NO: 23) R(Q/D)RFPY YFDYWGQGTLVTVSS

In some embodiments, an antibody of the invention comprises a V-segmentthat has at least 90% identity, or at least 91%, 92% 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identity to the germ-line segment VH 1-02 orVH1-03; or to one of the V-segments of the V_(H) regions shown in FIG.1, such as a V-segment portion of VH #1 (SEQ ID NO: 1), VH #2 (SEQ IDNO:2), VH #3 (SEQ ID NO:3), VH #4 (SEQ ID NO:4), or VH #5 (SEQ ID NO:5).

In some embodiments, the V-segment of the V_(H) region has a CDR1 and/orCDR2 as shown in FIG. 1. For example, an antibody of the invention mayhave a CDR1 that has the sequence GYYMH (SEQ ID NO: 24) or NYYIH (SEQ IDNO: 25); or a CDR2 that has the sequence WINPNSGGTNYAQKFQG (SEQ ID NO:26) or WINAGNGNTKYSQKFQG (SEQ ID NO: 27).

In particular embodiments, an antibody has both a CDR1 and a CDR2 fromone of the V_(H) region V-segments shown in FIG. 1 and a CDR3 thatcomprises R(Q/D)RFPY (SEQ ID NO: 22), e.g., RDRFPYYFDY (SEQ ID NO: 16)or RQRFPYYFDY (SEQ ID NO: 15). Thus, in some embodiments, an anti-GM-CSFantibody of the invention, may for example, have a CDR3-FR4 that has thesequence R(Q/D)RFPYYFDYWGQGTLVTVSS (SEQ ID NO: 23) and a CDR1 and/orCDR2 as shown in FIG. 1.

In some embodiments, a V_(H) region of an antibody of the invention hasa CDR3 that has a binding specificity determinant R(Q/D)RFPY (SEQ ID NO:22), a CDR2 from a human germline VH1 segment or a CDR1 from a humangermline VH1. In some embodiments, both the CDR1 and CDR2 are from humangermline VH1 segments.

III. Light Chains

A light chain of an anti-hGM-CSF antibody of the invention comprises atlight-chain V-region that comprises the following elements:

1) human light-chain V-segment sequences comprisingFR1-CDR1-FR2-CDR2-FR3

2) a CDRL3 region comprising the sequence FNK or FNR, e.g., QQFNRSPLT(SEQ ID NO: 28) or QQFNKSPLT (SEQ ID NO: 18).

3) a FR4 contributed by a human germ-line J-gene segment.

The V_(L) region comprises either a Vlambda or a Vkappa V-segment. Anexample of a Vkappa sequence that supports binding in combination with acomplementary V_(H)-region is provided in FIG. 1.

The V_(L) region CDR3 sequence comprises a J-segment derived sequence.In typical embodiments, the J-segment sequences in CDRL3 are from thesame J-segment used for FR4. Thus, the sequence in some embodiments maydiffer by not more than 2 amino acids from human kappa germ-lineV-segment and J-segment sequences. In some embodiments, the CDRL3-FR4region comprises the BSD and the complete human JK4 germline genesegment. Exemplary CDRL3-FR4 combinations for kappa chains are shownbelow in which the minimal essential binding specificity determinant isshown in bold and JK4 sequences are underlined:

CDR3 (SEQ ID NO: 29) QQFNRSPLTFGGGTKVEIK (SEQ ID NO: 30)QQFNKSPLTFGGGTKVEIK

The Vkappa segments are typically of the V KIII sub-class. In someembodiments, the segments have at least 80% sequence identity to a humangermline VKIII subclass, e.g., at least 80% identity to the humangerm-line VKIIIA27 (SEQ ID NO: 21) sequence. In some embodiments, theVkappa segment may differ by not more than 18 residues from VKIIIA27(SEQ ID NO: 21). In other embodiments, the VL region V-segment of anantibody of the invention has at least 85% identity, or at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to thehuman kappa V-segment sequence of a V_(L) region shown in FIG. 1, forexample, the V-segment sequence of VK #1 (SEQ ID NO: 6), VK #2 (SEQ IDNO: 7), VK #3 (SEQ ID NO: 8), or VK #4 (SEQ ID NO: 9).

In some embodiments, the variable region is comprised of human V-genesequences. For example, a variable region sequence can have at least 80%identity, or at least 85% identity, at least 90% identity, at least 95%identity, at least 96% identity, at least 97% identity, at least 98%identity, or at least 99% identity, or greater, with a human germ-lineV-gene sequence.

In some embodiments, the V-segment of the VL region has a CDR1 and/orCDR2 as shown in FIG. 1. For example, an antibody of the invention mayhave a CDR1 sequence of RASQSVGTNVA (SEQ ID NO: 31) or RASQSIGSNLA (SEQID NO: 32) RASQS(V/I)G(T/S)N(V/L)A (SEQ ID NO: 39); or a CDR2 sequenceSTSSRAT (SEQ ID NO: 33).

In particular embodiments, an anti-GM-CSF antibody of the invention mayhave a CDR1 and a CDR2 in a combination as shown in one of theV-segments of the VL regions set forth in FIG. 1 and a CDR3 sequencethat comprises FNK or FNR, e.g., the CDR3 may be QQFNKSPLT (SEQ ID NO:18) or QQFNRSPLT (SEQ ID NO: 28). In some embodiments, such a GM-CSFantibody may comprise an FR4 region that is FGGGTKVEIK (SEQ ID NO: 34).Thus, an anti-GM-CSF antibody of the invention, can comprise, e.g., boththe CDR1 and CDR2 from one of the VL regions shown in FIG. 1 and aCDR3-FR4 region that is FGGGTKVEIK (SEQ ID NO: 34).

IV. Preparation of hGM-CSF Antibodies

An antibody of the invention may comprise any of the V_(H) regions VH #1(SEQ ID NO: 1), VH #2 (SEQ ID NO: 2), VH #3 (SEQ ID NO: 3), VH #4 (SEQID NO: 4), or VH #5 (SEQ ID NO: 5) as shown in FIG. 1. In someembodiment, an antibody of the invention may comprise any of the V_(L)regions VK #1 (SEQ ID NO: 6), VK #2 (SEQ ID NO: 7), VK #3 (SEQ ID NO:8), or VK #4 (SEQ ID NO: 9) as shown in FIG. 1. In some embodiments, theantibody has a V_(H) region VH #1 (SEQ ID NO: 1), VH #2 (SEQ ID NO: 2),VH #3 (SEQ ID NO: 3), VH #4 (SEQ ID NO: 4), or VH #5 (SEQ ID NO: 5) asshown in FIG. 1; and a VL region VK #1 (SEQ ID NO: 6), VK #2 (SEQ ID NO:7), VK #3 (SEQ ID NO: 8), or VK #4 (SEQ ID NO: 9) as shown in FIG. 1, asdescribed, e.g., in U.S. Pat. Nos. 8,168,183 and 9,017,674, each ofwhich is incorporated herein by reference in its entirety.

An antibody may be tested to confirm that the antibody retains theactivity of antagonizing hGM-CSF activity. The antagonist activity canbe determined using any number of endpoints, including proliferationassays. Neutralizing antibodies and other hGM-CSF antagonists may beidentified or evaluated using any number of assays that assess hGM-CSFfunction. For example, cell-based assays for hGM-CSF receptor signaling,such as assays which determine the rate of proliferation of ahGM-CSF-dependent cell line in response to a limiting amount of hGM-CSF,are conveniently used. The human TF-1 cell line is suitable for use insuch an assay. See, Krinner et al., (2007) Mol. Immunol. In someembodiments, the neutralizing antibodies of the invention inhibithGM-CSF stimulated TF-I cell proliferation by at least 50%, when ahGM-CSF concentration is used which stimulates 90% maximal TF-I cellproliferation. Thus, typically, a neutralizing antibody, or otherhGM-CSF antagonist for use in the invention, has an EC50 of less than 10nM (e.g., Table 2). Additional assays suitable for use in identifyingneutralizing antibodies suitable for use with the present invention willbe well known to persons of skill in the art. In other embodiments, theneutralizing antibodies inhibit hGM-CSF stimulated proliferation by atleast about 75%, 80%, 90%, 95%, or 100%, of the antagonist activity ofthe antibody chimeric cl9/2, e.g., WO03/068920, which has the variableregions of the mouse monoclonal antibody LMM102 and the CDRs.

An exemplary chimeric antibody suitable for use as a hGM-CSF antagonistis cl9/2. The c 19/2 antibody binds hGM-CSF with a monovalent bindingaffinity of about 10 pM as determined by surface plasmon resonanceanalysis. The heavy and light chain variable region sequences of cl9/2are known (e.g., WO03/068920). The CDRs, as defined according to Kabat,are:

CDRH1 (SEQ ID NO: 35) DYNIH CDRH2 (SEQ ID NO: 36) YIAPYSGGTGYNQEFKNCDRH3 (SEQ ID NO: 16) RDRFPYYFDY CDRL1 (SEQ ID NO: 37) KASQNVGSNVA CDRL2(SEQ ID NO: 38) SASYRSG CDRL3 (SEQ ID NO: 28) QQFNRSPLT.

The CDRs can also be determined using other well-known definitions inthe art, e.g., Chothia, international ImMunoGeneTics database (IMGT),and AbM.

In some embodiments, an antibody used in the invention competes forbinding to, or binds to, the same epitope as cl9/2. The GM-CSF epitoperecognized by cl9/2 has been identified as a product that has twopeptides, residues 86-93 and residues 112-127, linked by a disulfidebond between residues 88 and 121. The cl9/2 antibody inhibits theGM-CSF-dependent proliferation of a human TF-I leukemia cell line withan EC50 of 30 pM when the cells are stimulated with 0.5 ng/ml GM-CSF. Insome embodiments, the antibody used in the invention binds to the sameepitope as cl9/2.

An antibody for administration, such as cl9/2, can be additionallyHumaneered. For example, the cl9/2 antibody can be further engineered tocontain human V gene segments.

A high-affinity antibody may be identified using well known assays todetermine binding activity and affinity. Such techniques include ELISAassays as well as binding determinations that employ surface plasmonresonance or interferometry. For example, affinities can be determinedby biolayer interferometry using a ForteBio (Mountain View, Calif.)Octet biosensor. An antibody of the invention typically binds withsimilar affinity to both glycosylated and non-glycosylated form ofhGM-CSF.

Antibodies of the invention compete with cl9/2 for binding to hGM-CSF.The ability of an antibody described herein to block or compete withcl9/2 for binding to hGM-CSF indicates that the antibody binds to thesame epitope cl9/2 or to an epitope that is close to, e.g., overlapping,with the epitope that is bound by cl9/2. In other embodiments anantibody described herein, e.g., an antibody comprising a V_(H) andV_(L) region combination as shown in the table provided in FIG. 1, canbe used as a reference antibody for assessing whether another antibodycompetes for binding to hGM-CSF. A test antibody is considered tocompetitively inhibit binding of a reference antibody, if binding of thereference antibody to the antigen is reduced by at least 30%, usually atleast about 40%, 50%, 60% or 75%, and often by at least about 90%, inthe presence of the test antibody. Many assays can be employed to assessbinding, including ELISA, as well as other assays, such as immunoblots.In some embodiments, an antibody of the invention has a dissociationrate that is at least 2 to 3-fold slower than a reference chimeric cl9/2monoclonal antibody assayed under the same conditions, but has a potencythat is at least 6-10 times greater than that of the reference antibodyin neutralizing hGM-CSF activity in a cell-based assay that measureshGM-CSF activity.

Methods for the isolation of antibodies with V-region sequences close tohuman germ-line sequences have previously been described (US patentapplication publication nos. 20050255552 and 20060134098). Antibodylibraries may be expressed in a suitable host cell including mammaliancells, yeast cells or prokaryotic cells. For expression in some cellsystems, a signal peptide can be introduced at the N-terminus to directsecretion to the extracellular medium. Antibodies may be secreted frombacterial cells such as E. coli with or without a signal peptide.Methods for signal-less secretion of antibody fragments from E. coli aredescribed in US patent application 20070020685.

In some embodiments, an hGM-CSF-binding antibody of the invention isgenerated where, an antibody that has a CDR from one of the VH-regionsof the invention shown in FIG. 1, is combined with an antibody having aCDR of one of the V_(L)-regions shown in FIG. 1, and expressed in any ofa number of formats in a suitable expression system. Thus, the antibodymay be expressed as a scFv, Fab, Fab′ (containing an immunoglobulinhinge sequence), F(ab′)₂, (formed by di-sulfide bond formation betweenthe hinge sequences of two Fab′ molecules), whole immunoglobulin ortruncated immunoglobulin or as a fusion protein in a prokaryotic oreukaryotic host cell, either inside the host cell or by secretion. Amethionine residue may optionally be present at the N-terminus, forexample, in polypeptides produced in signal-less expression systems.Each of the V_(H)-regions described herein may be paired with each ofthe VL regions to generate an anti-hGM-CSF antibody. In an embodiment, afusion protein comprises an anti-hGM-CSF-binding antibody of theinvention or a fragment thereof (in non-limiting examples, ananti-hGM-CSF antibody fragment is a Fab, a Fab′, a F(ab′)2, a scFv, or adAB), and human transferrin, wherein the human transferrin is fused tothe antibody at the end of the heavy chain constant region 1 (C_(H)1),after the hinge, or after C_(H)3, as described in Shin, S-U., et al.Proc. Natl. Acad. Sci. USA, Vol. 92, pp. 2820-2824, 1995, which isincorporated herein by reference in its entirety.

Exemplary combinations of heavy and light chains are shown in the tableprovided in FIG. 1. In some embodiment, the antibody VL region, e.g., VK#1 (SEQ ID NO: 6), VK #2 (SEQ ID NO: 7), VK #3 (SEQ ID NO: 8), or VK #4(SEQ ID NO: 9) of FIG. 1, is combined with a human kappa constant regionto form the complete light-chain. Further, in some embodiments, the VHregion is combined a human gamma-1 constant regions. Any suitablegamma-1 allotype can be chose, such as the f-allotype. Thus, in someembodiments, the antibody is an IgG, e.g., having an f-allotype, thathas a VH selected from VH #1 (SEQ ID NO:1), VH #2 (SEQ ID NO:2), VH #3(SEQ ID NO:3), VH #4 (SEQ ID NO:4), or VH #5 (SEQ ID NO:5) (FIG. 1), anda VL selected from VK #1 (SEQ ID NO: 6), VK #2 (SEQ ID NO: 7), VK #3(SEQ ID NO: 8), or VK #4 (SEQ ID NO: 9) (FIG. 1).

The antibodies of the invention inhibit hGM-CSF receptor activation,e.g., by inhibiting hGM-CSF binding to the receptor, and exhibit highaffinity binding to hGM-CSF, e.g., 500 pM. In some embodiments, theantibody has a dissociation constant of about 10⁻⁴ per sec or less. Notto be bound by theory, an antibody with a slower dissociation constantprovides improved therapeutic benefit. For example, an antibody of theinvention that has a three-fold slower off-rate than cl9/2, produced a10-fold more potent hGM-CSF neutralizing activity, e.g., in a cell-basedassay such as IL-8 production (see, e.g., Example 2).

Antibodies may be produced using any number of expression systems,including both prokaryotic and eukaryotic expression systems. In someembodiments, the expression system is a mammalian cell expression, suchas a CHO cell expression system. Many such systems are widely availablefrom commercial suppliers. In embodiments in which an antibody comprisesboth a V_(H) and V_(L) region, the V_(H) and V_(L) regions may beexpressed using a single vector, e.g., in a dicistronic expression unit,or under the control of different promoters. In other embodiments, theV_(H) and V_(L) region may be expressed using separate vectors. A V_(H)or V_(L) region as described herein may optionally comprise a methionineat the N-terminus.

An antibody of the invention may be produced in any number of formats,including as a Fab, a Fab′, a F(ab′)₂, a scFv, or a dAB. An antibody ofthe invention can also include a human constant region. The constantregion of the light chain may be a human kappa or lambda constantregion. The heavy chain constant region is often a gamma chain constantregion, for example, a gamma-1, gamma-2, gamma-3, or gamma-4 constantregion. In other embodiments, the antibody may be an IgA.

In some embodiments of the invention, the antibody V_(L) region, e.g.,VK #1 (SEQ ID NO: 6), VK #2 (SEQ ID NO: 7), VK #3 (SEQ ID NO: 8), or VK#4 (SEQ ID NO: 9) of FIG. 1, is combined with a human kappa constantregion (e.g., SEQ ID NO:10) to form the complete light-chain.

In some embodiments of the invention, the V_(H) region is combined ahuman gamma-1 constant region. Any suitable gamma-1 f allotype can bechosen, such as the f-allotype. Thus, in some embodiments, the antibodyis an IgG having an f-allotype constant region, e.g., SEQ ID NO:11, thathas a V_(H) selected from VH #1 (SEQ ID NO:1), VH #2 (SEQ ID NO:2), VH#3 (SEQ ID NO:3), VH #4 (SEQ ID NO:4), or VH #5 (SEQ ID NO:5) (FIG. 1).In some embodiments, the antibody has a V_(L) selected from VK #1 (SEQID NO: 6), VK #2 (SEQ ID NO: 7), VK #3 (SEQ ID NO: 8), or VK #4 (SEQ IDNO: 9) (FIG. 1.) In particular embodiments, the antibody has a kappaconstant region as set forth in SEQ ID NO:10, and a heavy chain constantregion as set forth in SEQ ID NO:11, where the heavy and light chainvariable regions comprise one of the following combinations from thesequences set forth in FIG. 1: a) VH #2 (SEQ ID NO:2), VK #3 (SEQ IDNO:8); b) VH #1 (SEQ ID NO:1), VK #3 (SEQ ID NO:8); c) VH #3 (SEQ IDNO:3), VK #1 (SEQ ID NO:6); d) VH #3 (SEQ ID NO:3), VK #3 (SEQ ID NO:8);e) VH #4 (SEQ ID NO:4), VK #4 (SEQ ID NO:9); f) VH #4 (SEQ ID NO:4), VK#2 (SEQ ID NO:7); g) VH #5 (SEQ ID NO:5), VK #1 (SEQ ID NO:6); h) VH #5(SEQ ID NO:5), VK #2 (SEQ ID NO:7); i) VH #3 (SEQ ID NO:3), VK #4 (SEQID NO:9); or j) VH #3 (SEQ ID NO:3), VK #3 (SEQ ID NO:8).

In some embodiments, e.g., where the antibody is a fragment, theantibody can be conjugated to another molecule, e.g., polyethyleneglycol (PEGylation) or serum albumin, to provide an extended half-lifein vivo. Examples of PEGylation of antibody fragments are provided inKnight et al. Platelets 15:409, 2004 (for abciximab); Pedley et al., Br.J. Cancer 70:1126, 1994 (for an anti-CEA antibody); Chapman et al.,Nature Biotech. 17:780, 1999; and Humphreys, et al., Protein Eng. Des.20: 227, 2007).

In some embodiments, the antibodies of the invention are in the form ofa Fab′ fragment. A full-length light chain is generated by fusion of aV_(L)-region to human kappa or lambda constant region. Either constantregion may be used for any light chain; however, in typical embodiments,a kappa constant region is used in combination with a Vkappa variableregion and a lambda constant region is used with a Vlambda variableregion.

The heavy chain of the Fab′ is a Fd′ fragment generated by fusion of aV_(H)-region of the invention to human heavy chain constant regionsequences, the first constant (CH1) domain and hinge region. The heavychain constant region sequences can be from any of the immunoglobulinclasses, but is often from an IgG, and may be from an IgG1, IgG2, IgG3or IgG4. The Fab′ antibodies of the invention may also be hybridsequences, e.g., a hinge sequence may be from one immunoglobulinsub-class and the CH1 domain may be from a different sub-class.

V. Administration of Anti-hGM-CSF Antibodies for the Treatment ofDiseases in which GM-CSF is a Target.

The invention also provides methods of treating a patient that has adisease involving hGM-CSF in which it is desirable to inhibit hGM-CSFactivity, i.e., in which hGM-CSF is a therapeutic target. In someembodiments, such a patient has a chronic inflammatory disease, e.g.,arthritis, e.g., rheumatoid arthritis, psoriatic arthritis, ankylosingspondylitis, juvenile idiopathic arthritis, systemic-onset Still'sdisease and other inflammatory diseases of the joints; inflammatorybowel diseases, e.g., ulcerative colitis, Crohn's disease, Barrett'ssyndrome, ileitis, enteritis, eosinophilic esophagitis andgluten-sensitive enteropathy; inflammatory disorders of the respiratorysystem, such as asthma, eosinophilic asthma, adult respiratory distresssyndrome, allergic rhinitis, silicosis, chronic obstructive pulmonarydisease, hypersensitivity lung diseases, interstitial lung disease,diffuse parenchymal lung disease, bronchiectasis; inflammatory diseasesof the skin, including psoriasis, scleroderma, and inflammatorydermatoses such as eczema, atopic dermatitis, urticaria, and pruritis;disorders involving inflammation of the central and peripheral nervoussystem, including multiple sclerosis, idiopathic demyelinatingpolyneuropathy, Guillain-Barre syndrome, chronic inflammatorydemyelinating polyneuropathy, neurofibromatosis and neurodegenerativediseases such as Alzheimer's disease. Various other inflammatorydiseases can be treated using the methods of the invention. Theseinclude systemic lupus erythematosis, immune-mediated renal disease,e.g., glomerulonephritis, and spondyloarthropathies; and diseases withan undesirable chronic inflammatory component such as systemicsclerosis, idiopathic inflammatory myopathies, Sjogren's syndrome,vasculitis, sarcoidosis, thyroiditis, gout, otitis, conjunctivitis,sinusitis, sarcoidosis, Behcet's syndrome, autoimmunelymphoproliferative syndrome (or ALPS, also known as Canale-Smithsyndrome), Ras-associated autoimmune leukoproliferative disorder (orRALD), Noonan syndrome, hepatobiliary diseases such as hepatitis,primary biliary cirrhosis, granulomatous hepatitis, and sclerosingcholangitis. In some embodiments, the patient has inflammation followinginjury to the cardiovascular system. Various other inflammatory diseasesinclude Kawasaki's disease, Multicentric Castleman's Disease,tuberculosis and chronic cholecystitis. Additional chronic inflammatorydiseases are described, e.g., in Harrison's Principles of InternalMedicine, 12th Edition, Wilson, et al., eds., McGraw-Hill, Inc.). Insome embodiments, a patient treated with an antibody has a cancer inwhich GM-CSF contributes to tumor or cancer cell growth, including butnot limited to, e.g., acute myeloid leukemia, plexiformneurofibromatosis, autoimmune lymphoproliferative syndrome (or ALPS,also known as Canale-Smith syndrome), Ras-associated autoimmuneleukoproliferative disorder (or RALD), Noonan syndrome, chronicmyelomonocytic leukemia, juvenile myelomonocytic leukemia, and acutemyeloid leukemia. In some embodiments, a patient treated with anantibody of the invention has, or is at risk of heart failure, e.g., dueto ischemic injury to the cardiovascular system such as ischemic heartdisease, stroke, and atherosclerosis. In some embodiments, a patienttreated with an antibody of the invention has asthma. In someembodiments, a patient treated with an antibody of the invention hasAlzheimer's disease. In some embodiments, a patient treated with anantibody of the invention has osteopenia, e.g., osteoporosis. In someembodiments, a patient treated with an antibody of the invention hasthrombocytopenia purpura. In some embodiments, the patient has Type I orType II diabetes. In some embodiments, a patient may have more than onedisease in which GM-CSF is a therapeutic target, e.g., a patient mayhave rheumatoid arthritis and heart failure, or osteoporosis andrheumatoid arthritis, etc.

Two other examples of neutralizing anti-GM-CSF antibody are the humanElO antibody and human G9 antibody described in Li et al, (2006) PNAS103(10):3557-3562. ElO and G9 are IgG class antibodies. ElO has an 870pM binding affinity for GM-CSF and G9 has a 14 pM affinity for GM-CSF.Both antibodies are specific for binding to human GM-CSF and show strongneutralizing activity as assessed with a TFl cell proliferation assay.

An additional exemplary neutralizing anti-GM-CSF antibody is the MT203antibody described by Krinner et al, (Mol Immunol. 44:916-25, 2007; Epub2006 May 112006). MT203 is an IgG1 class antibody that binds GM-CSF withpicomolar affinity. The antibody shows potent inhibitory activity asassessed by TF-I cell proliferation assay and its ability to block IL-8production in U937 cells.

Additional antibodies suitable for use with the present invention willbe known to persons of skill in the art.

hGM-CSF antagonists that are anti-hGM-CSF receptor antibodies can alsobe employed with the methods of the present disclosure. Such hGM-CSFantagonists include antibodies to the hGM-CSF receptor alpha chain orbeta chain. An anti-hGM-CSF receptor antibody employed in the inventioncan be in any antibody format as explained above, e.g., intact,chimeric, monoclonal, polyclonal, antibody fragment, humanized,Humaneered, and the like. Examples of anti-hGM-CSF receptor antibodies,e.g., neutralizing, high-affinity antibodies, suitable for use in theinvention are known (see, e.g., U.S. Pat. No. 5,747,032 and Nicola etal., Blood 82: 1724, 1993).

Non-Antibody GM-CSF Antagonists

Other proteins that may interfere with the productive interaction ofhGM-CSF with its receptor include mutant hGM-CSF proteins and secretedproteins comprising at least part of the extracellular portion of one orboth of the hGM-CSF receptor chains that bind to hGM-CSF and competewith binding to cell-surface receptor. For example, a soluble hGM-CSFreceptor antagonist can be prepared by fusing the coding region of thesGM-CSFR alpha with the CH2-CH3 regions of murine IgG2a. An exemplarysoluble hGM-CSF receptor is described by Raines et al. (1991) Proc.Natl. Acad. Sci USA 88: 8203. An example of a GM-CSFR alpha-Fc fusionprotein is provided, e.g., in Brown et al (1995) Blood 85: 1488. In someembodiments, the Fc component of such a fusion can be engineered tomodulate binding, e.g., to increase binding, to the Fc receptor.

Other hGM-CSF antagonists include hGM-CSF mutants. For example, hGM-CSFhaving a mutation of amino acid residue 21 of hGM-CSF to Arginine orLysine (E21R or E21K) described by Hercus et al. Proc. Natl. Acad. SciUSA 91:5838, 1994 has been shown to have in vivo activity in preventingdissemination of hGM-CSF-dependent leukemia cells in mouse xenograftmodels (Iversen et al. Blood 90:4910, 1997). As appreciated by one ofskill in the art, such antagonists can include conservatively modifiedvariants of hGM-CSF that have substitutions, such as the substitutionnoted at amino acid residue 21, or hGM-CSF variants that have, e.g.,amino acid analogs to prolong half-life.

In some embodiments, the hGM-CSF antagonist may be a peptide. Forexample, an hGM-CSF peptide antagonist may be a peptide designed tostructurally mimic the positions of specific residues on the B and Chelices of human GM-CSF that are implicated in receptor binding andbioactivity (e.g., Monfardini et al, J. Biol. Chem 271:2966-2971, 1996).

In other embodiments, the hGM-CSF antagonist is an “antibody mimetic”that targets and binds to the antigen in a manner similar to antibodies.Certain of these “antibody mimics” use non-immunoglobulin proteinscaffolds as alternative protein frameworks for the variable regions ofantibodies. For example, Ku et al. (Proc. Natl. Acad. Sci. U.S.A.92(14):6552-6556 (1995)) discloses an alternative to antibodies based oncytochrome b562 in which two of the loops of cytochrome b562 wererandomized and selected for binding against bovine serum albumin. Theindividual mutants were found to bind selectively with BSA similarlywith anti-BSA antibodies. U.S. Pat. Nos. 6,818,418 and 7,115,396disclose an antibody mimic featuring a fibronectin or fibronectin-likeprotein scaffold and at least one variable loop. Known as Adnectins,these fibronectin-based antibody mimics exhibit many of the samecharacteristics of natural or engineered antibodies, including highaffinity and specificity for any targeted ligand. The structure of thesefibronectin-based antibody mimics is similar to the structure of thevariable region of the IgG heavy chain. Therefore, these mimics displayantigen binding properties similar in nature and affinity to those ofnative antibodies. Further, these fibronectin-based antibody mimicsexhibit certain benefits over antibodies and antibody fragments. Forexample, these antibody mimics do not rely on disulfide bonds for nativefold stability, and are, therefore, stable under conditions which wouldnormally break down antibodies. In addition, since the structure ofthese fibronectin-based antibody mimics is similar to that of the IgGheavy chain, the process for loop randomization and shuffling may beemployed in vitro that is similar to the process of affinity maturationof antibodies in vivo.

Beste et al. (Proc. Natl. Acad. Sci. U.S.A. 96(5):1898-1903 (1999))disclose an antibody mimic based on a lipocalin scaffold (Anticalin®).Lipocalins are composed of a β-barrel with four hypervariable loops atthe terminus of the protein. The loops were subjected to randommutagenesis and selected for binding with, for example, fluorescein.Three variants exhibited specific binding with fluorescein, with onevariant showing binding similar to that of an anti-fluorescein antibody.Further analysis revealed that all of the randomized positions arevariable, indicating that Anticalin would be suitable to be used as analternative to antibodies. Thus, Anticalins are small, single chainpeptides, typically between 160 and 180 residues, which provides severaladvantages over antibodies, including decreased cost of production,increased stability in storage and decreased immunological reaction.

U.S. Pat. No. 5,770,380 discloses a synthetic antibody mimetic using therigid, non-peptide organic scaffold of calixarene, attached withmultiple variable peptide loops used as binding sites. The peptide loopsall project from the same side geometrically from the calixarene, withrespect to each other. Because of this geometric confirmation, all ofthe loops are available for binding, increasing the binding affinity toa ligand. However, in comparison to other antibody mimics, thecalixarene-based antibody mimic does not consist exclusively of apeptide, and therefore it is less vulnerable to attack by proteaseenzymes. Neither does the scaffold consist purely of a peptide, DNA orRNA, meaning this antibody mimic is relatively stable in extremeenvironmental conditions and has a long life-span. Further, since thecalixarene-based antibody mimic is relatively small, it is less likelyto produce an immunogenic response.

Murali et al. (Cell Mol Biol 49(2):209-216 (2003)) describe amethodology for reducing antibodies into smaller peptidomimetics, theyterm “antibody-like binding peptidomimetics” (ABiP) which may also beuseful as an alternative to antibodies.

In addition to non-immunoglobulin protein frameworks, antibodyproperties have also been mimicked in compounds comprising RNA moleculesand unnatural oligomers (e.g., protease inhibitors, benzodiazepines,purine derivatives and beta-turn mimics). Accordingly, non-antibodyGM-CSF antagonists can also include such compounds.

Therapeutic Administration

In some embodiments, the methods of the present disclosure compriseadministering a hGM-CSF antagonist, (e.g., an anti-hGM-CSF antibody) asa pharmaceutical composition to a subject having a CRS or a cytokinestorm. In some embodiments, the hGM-CSF antagonist is administered in atherapeutically effective amount using a dosing regimen suitable fortreatment of the disease.

In some embodiments, a therapeutically effective amount is an amountthat at least partially arrests the condition or its symptoms. Forexample, a therapeutically effective amount may arrest immuneactivation, may decrease the levels of circulating cytokines, maydecrease T-cell activation, or may ameliorate fever, malaise, fatigue,anorexia, myalgias, arthalgias, nausea, vomiting, headache, skin rash,nausea, vomiting, diarrhea, tachypnea, hypoxemia, cardiovasculartachycardia, widened pulse pressure, hypotension, increased cardiacoutput (early), potentially diminished cardiac output (late), elevatedD-dimer, hypofibrinogenemia with or without bleeding, azotemia,transaminitis, hyperbilirubinemia, headache, mental status changes,confusion, delirium, word finding difficulty or frank aphasia,hallucinations, tremor, dysmetria, altered gait, or seizures.

The methods of the invention comprise administering an anti-hGM-CSFantibody as a pharmaceutical composition to a patient in atherapeutically effective amount using a dosing regimen suitable fortreatment of the disease. The composition can be formulated for use in avariety of drug delivery systems. One or more physiologically acceptableexcipients or carriers can also be included in the compositions forproper formulation. Suitable formulations for use in the presentinvention are found in Remington: The Science and Practice of Pharmacy,21st Edition, Philadelphia, Pa. Lippincott Williams & Wilkins, 2005. Fora brief review of methods for drug delivery, see, Langer, Science 249:1527-1533 (1990).

The anti-hGM-CSF antibody for use in the methods of the invention isprovided in a solution suitable for injection into the patient such as asterile isotonic aqueous solution for injection. The antibody isdissolved or suspended at a suitable concentration in an acceptablecarrier. In some embodiments the carrier is aqueous, e.g., water,saline, phosphate buffered saline, and the like. The compositions maycontain auxiliary pharmaceutical substances as required to approximatephysiological conditions, such as pH adjusting and buffering agents,tonicity adjusting agents, and the like.

The pharmaceutical compositions of the invention are administered to apatient, e.g., a patient that has osteopenia, rheumatoid arthritis,juvenile idiopathic arthritis, systemic-onset Still's disease, asthma,eosinophilic asthma, eosinophilic esophagitis, multiple sclerosis,psoriasis, atopic dermatitis, plexiform neurofibromatosis, autoimmunelymphoproliferative syndrome (or ALPS, also known as Canale-Smithsyndrome), Ras-associated autoimmune leukoproliferative disorder (orRALD), Noonan syndrome, chronic myelomonocytic leukemia, juvenilemyelomonocytic leukemia, acute myeloid leukemia, MulticentricCastleman's Disease, chronic obstructive pulmonary disease, interstitiallung disease, diffuse parenchymal lung disease, idiopathicthrombocytopenia purpura, Alzheimer's disease, heart failure, Kawasaki'sDisease, cardiac damage due to an ischemic event, or diabetes, in anamount sufficient to cure or at least partially arrest the disease orsymptoms of the disease and its complications. An amount adequate toaccomplish this is defined as a “therapeutically effective dose.” Atherapeutically effective dose is determined by monitoring a patient'sresponse to therapy. Typical benchmarks indicative of a therapeuticallyeffective dose includes amelioration of symptoms of the disease in thepatient. Amounts effective for this use will depend upon the severity ofthe disease and the general state of the patient's health, includingother factors such as age, weight, gender, administration route, etc.Single or multiple administrations of the antibody may be administereddepending on the dosage and frequency as required and tolerated by thepatient. In any event, the methods provide a sufficient quantity ofanti-hGM-CSF antibody to effectively treat the patient.

The antibody may be administered alone, or in combination with othertherapies to treat the disease of interest.

The antibody can be administered by injection or infusion through anysuitable route including but not limited to intravenous, sub-cutaneous,intramuscular or intraperitoneal routes. In some embodiments, theantibody may be administered by insufflation. In an exemplaryembodiment, the antibody may be stored at 10 mg/ml in sterile isotonicaqueous saline solution for injection at 4° C. and is diluted in either100 ml or 200 ml 0.9% sodium chloride for injection prior toadministration to the patient. The antibody is administered byintravenous infusion over the course of 1 hour at a dose of between 0.2and 10 mg/kg. In other embodiments, the antibody is administered, forexample, by intravenous infusion over a period of between 15 minutes and2 hours. In still other embodiments, the administration procedure is viasub-cutaneous or intramuscular injection.

In some embodiments, the hGM-CSF antagonist, e.g., an anti-hGM-CSFantibody, is administered by a perispinal route. Perispinaladministration involves anatomically localized delivery performed so asto place the therapeutic molecule directly in the vicinity of the spineat the time of initial administration. Perispinal administration isdescribed, e.g., in U.S. Pat. No. 7,214,658 and in Tobinick & Gross, J.Neuroinflammation 5:2, 2008.

The dose of hGM-CSF antagonist is chosen in order to provide effectivetherapy for a subject that has been diagnosed with CRS or cytokinestorm. The dose is typically in the range of about 0.1 mg/kg body weightto about 50 mg/kg body weight or in the range of about 1 mg to about 2 gper patient. The dose is often in the range of about 1 to about 20 mg/kgor approximately about 50 mg to about 2000 mg/patient. The dose may berepeated at an appropriate frequency which may be in the range once perday to once every three months, depending on the pharmacokinetics of theantagonist (e.g. half-life of the antibody in the circulation) and thepharmacodynamic response (e.g. the duration of the therapeutic effect ofthe antibody). In some embodiments where the antagonist is an antibodyor modified antibody fragment, the in vivo half-life of between about 7and about 25 days and antibody dosing is repeated between once per weekand once every 3 months. In other embodiments, the antibody isadministered approximately once per month.

A V_(H) region and/or V_(L) region of the invention may also be used fordiagnostic purposes. For example, the V_(H) and/or V_(L) region may beused for clinical analysis, such as detection of GM-CSF levels in apatient. A V_(H) or V_(L) region of the invention may also be used,e.g., to produce anti-Id antibodies.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present application shall have the meanings that arecommonly understood by those of skill in the art. Further, unlessotherwise required by context, singular terms shall include pluralitiesand plural terms shall include the singular.

In one embodiment, “treating” comprises therapeutic treatment and“preventing” comprises prophylactic or preventative measures, whereinthe object is to prevent or lessen the targeted pathologic condition ordisorder as described hereinabove. Thus, in one embodiment, treating mayinclude directly affecting or curing, suppressing, inhibiting,preventing, reducing the severity of, delaying the onset of, reducingsymptoms associated with the disease, disorder or condition, or acombination thereof. Thus, in one embodiment, “treating,”“ameliorating,” and “alleviating” refer inter alia to delayingprogression, expediting remission, inducing remission, augmentingremission, speeding recovery, increasing efficacy of or decreasingresistance to alternative therapeutics, or a combination thereof. In oneembodiment, “preventing” refers, inter alia, to delaying the onset ofsymptoms, preventing relapse to a disease, decreasing the number orfrequency of relapse episodes, increasing latency between symptomaticepisodes, or a combination thereof. In one embodiment, “suppressing” or“inhibiting”, refers inter alia to reducing the severity of symptoms,reducing the severity of an acute episode, reducing the number ofsymptoms, reducing the incidence of disease-related symptoms, reducingthe latency of symptoms, ameliorating symptoms, reducing secondarysymptoms, reducing secondary infections, prolonging patient survival, ora combination thereof.

In the present disclosure the singular forms “a,” “an,” and “the”include the plural reference, and reference to a particular numericalvalue includes at least that particular value, unless the contextclearly indicates otherwise. The term “plurality”, as used herein, meansmore than one. When a range of values is expressed, another embodimentincludes from the one particular and/or to the other particular value.

Similarly, when values are expressed as approximations, by use of theantecedent “about,” it is understood that the particular value formsanother embodiment. All ranges are inclusive and combinable. In someembodiments, the term “about”, refers to a deviance of between 0.0001-5%from the indicated number or range of numbers. In some embodiments, theterm “about”, refers to a deviance of between 1-10% from the indicatednumber or range of numbers. In some embodiments, the term “about”,refers to a deviance of up to 25% from the indicated number or range ofnumbers. The term “comprises” means encompasses all the elements listed,but may also include additional, unnamed elements, and it may be usedinterchangeably with the terms “encompasses”, “includes”, or “contains”having all the same qualities and meanings. The term “consisting of”means being composed of the recited elements or steps, and it may beused interchangeably with the terms “composed of” having all the samequalities and meanings.

EXAMPLES Example 1—Exemplary Humaneered Antibodies to GM-CSF

A panel of engineered Fab′ molecules with the specificity of cl9/2 weregenerated from epitope-focused human V-segment libraries as described inUS patent application publication nos. 20060134098 and 20050255552.Epitope-focused libraries were constructed from human V-segment librarysequences linked to a CDR3-FR4 region containing BSD sequences in CDRH3and CDRL3 together with human germ-line J-segment sequences. For theheavy chain, human germ-line JH4 sequence was used and for the lightchain, human germ-line JK4 sequence was used.

Full-length Humaneered V-regions from a Vh1-restricted library wereselected that supported binding to recombinant human GM-CSF. The“full-length” V-kappa library was used as a base for construction of“cassette” libraries as described in US patent application publicationno. 20060134098, in which only part of the murine cl9/2 V-segment wasinitially replaced by a library of human sequences. Two types ofcassettes were constructed. Cassettes for the V-kappa chains were madeby bridge PCR with overlapping common sequences within the framework 2region. In this way “front-end” and “middle” human cassette librarieswere constructed for the human V-kappa III isotype. Human V-kappa IIIcassettes which supported binding to GM-CSF were identified bycolony-lift binding assay and ranked according to affinity in ELISA. TheV-kappa human “front-end” and “middle” cassettes were fused together bybridge PCR to reconstruct a fully human V-kappa region that supportedGM-CSF binding activity. The Humaneered Fibs thus consist of HumaneeredV-heavy and V-kappa regions that support binding to human GM-CSF.

Binding activity was determined by surface plasmon resonance (spr)analysis. Biotinylated GM-CSF was captured on a streptavidin-coated CM5biosensor chip. Humaneered Fab fragments expressed from E. coli werediluted to a starting concentration of 30 nM in 10 mM HEPES, 150 mMNaCl, 0.1 mg/ml BSA and 0.005% P20 at pH 7.4. Each Fab was diluted 4times using a 3-fold dilution series and each concentration was testedtwice at 37 degrees C. to determine the binding kinetics with thedifferent density antigen surfaces. The data from all three surfaceswere fit globally to extract the dissociation constants.

Binding kinetics were analyzed by Biacore 3000 surface plasmon resonance(SPR). Recombinant human GM-CSF antigen was biotinylated and immobilizedon a streptavidin CM5 sensor chip. Fab samples were diluted to astarting concentration of 3 nM and run in a 3-fold dilution series.Assays were run in 10 mM HEPES, 150 mM NaCl, 0.1 mg/mL BSA and 0.005%p20 at pH 7.4 and 37° C. Each concentration was tested twice. Fab′binding assays were run on two antigen density surfaces providingduplicate data sets. The mean affinity (KD) for each of 6 varioushumaneered anti-GM-CSF Fab clones, calculated using a 1: 1 Langmuirbinding model, is shown in Table 2.

Fabs were tested for GM-CSF neutralization using a TF-I cellproliferation assay. GM-CSF-dependent proliferation of human TF-I cellswas measured after incubation for 4 days with 0.5 ng/ml GM-CSF using aMTS assay (Cell titer 96, Promega) to determine viable cells. All Fabsinhibited cell proliferation in this assay indicating that these areneutralizing antibodies. There is a good correlation between relativeaffinities of the anti-GM-CSF Fabs and EC50 in the cell-based assay.Anti-GM-CSF antibodies with monovalent affinities in the range 18 pM-104pM demonstrate effective neutralization of GM-CSF in the cell-basedassay.

Exemplary engineered anti-GM-CSF V region sequences are shown in FIG. 1.

TABLE 2 Affinity of anti-GM-CSF Fabs determined by surface plasmonresonance analysis in comparison with activity (EC50) in a GM-CSFdependent TF-I cell proliferation assay Monovalent EC₅₀ (pM) in TF-binding affinity 1 cell determined by proliferation Fab SPR (pM) assay94 18 165 104 19 239 77 29 404 92 58 539 42 104 3200 44 81 7000

Example 2—Evaluation of a Humaneered GM-CSF Antibody

This example evaluates the binding activity and biological potency of ahumaneered anti-GM-CSF antibody in a cell-based assay in comparison to achimeric IgG1k antibody (Ab2) having variable regions from the mouseantibody LMM102 (Nice et al., Growth Factors 3:159, 1990). Ab1 is ahumaneered IgG1k antibody against GM-CSF having identical constantregions to Ab2.

Surface Plasmon Resonance Analysis of Binding of Human GM-CSF to Ab1 andAb2

Surface Plasmon resonance analysis was used to compare binding kineticsand monovalent affinities for the interaction of Ab1 and Ab2 withglycosylated human GM-CSF using a Biacore 3000 instrument. Ab1 or Ab2was captured onto the Biacore chip surface using polyclonal anti-humanF(ab′)2. Glycosylated recombinant human GM-CSF expressed from human 293cells was used as the analyte. Kinetic constants were determined in 2independent experiments (see FIGS. 2A-2B and Table 3). The results showthat GM-CSF bound to Ab2 and Ab1 with comparable monovalent affinity inthis experiment. However, Ab1 had a two-fold slower “on-rate” than Ab2,but an “off-rate” that was approximately three-fold slower.

TABLE 3 Kinetic constants at 37° C. determined from the surface plasmonresonance analysis in FIGS. 2A-2B; association constant (k_(a)),dissociation constant (k_(d)) and calculated affinity (KD) are shown.k_(a) (M⁻¹s⁻¹) k_(d) (s⁻¹) KD (pM) Ab2 7.20 × 10⁵  2.2 × 10⁻⁵ 30.5 Ab12.86 × 10⁵ 7.20 × 10⁻⁶ 25.1

GM-CSF is naturally glycosylated at both N-linked and O-linkedglycosylation sites although glycosylation is not required forbiological activity. In order to determine whether GM-CSF glycosylationaffects the binding of Ab1 or Ab2, the antibodies were compared in anELISA using recombinant GM-CSF from two different sources; GM-CSFexpressed in E. coli (non-glycosylated) and GM-CSF expressed from human293 cells (glycosylated). The results in FIGS. 3A-3B and Table 4 showedthat both antibodies bound glycosylated and non-glycosylated GM-CSF withequivalent activities. The two antibodies also demonstrated comparableEC₅₀ values in this assay.

TABLE 4 Summary of EC₅₀ for binding of Ab2 and Ab1 to human GM-CSF fromtwo different sources determined by ELISA. Binding to recombinant GM-CSFfrom human 293 cells (glycosylated) or from E. coli (non-glycosylated)was determined from two independent experiments. Experiment 1 is shownin FIGS. 3A-3B. Non-glycosylated Non-glycosylated Glycosylated (exp 1)(exp 2) (exp 1) Ab2 400 pM 433 pM 387 pM Ab1 373 pM 440 pM 413 pM

Ab1 is a Humaneered antibody that was derived from the mouse variableregions present in Ab2. Ab1 was tested for overlapping epitopespecificity (Ab2) by competition ELISA.

Biotinylated Ab2 was prepared using known techniques. Biotinylation didnot affect binding of Ab2 to GM-CSF as determined by ELISA. In theassay, Ab2 or Ab1 was added in varying concentrations with a fixedamount of biotinylated Ab2. Detection of biotinylated Ab2 was assayed inthe presence of unlabeled Ab or Ab1 competitor (FIGS. 4A-4B). Both Ab1and Ab2 competed with biotinylated Ab2 for binding to GM-CSF, thusindicating binding to the same epitope. Ab1 competed more effectivelyfor binding to GM-CSF than Ab2, consistent with the slower dissociationkinetics for Ab1 when compared with Ab2 by surface plasmon resonanceanalysis.

Neutralization of GM-CSF Activity by Ab1 and Ab2

A cell-based assay for neutralization of GM-CSF activity was employed toevaluate biological potency. The assay measures IL-8 secretion from U937cells induced with GM-CSF. IL-8 secreted into the culture supernatant isdetermined by ELISA after 16 hours induction with 0.5 ng/ml E.coli-derived GM-CSF.

A comparison of the neutralizing activity of Ab1 and Ab2 in this assayis shown in a representative assay in FIG. 5. In three independentexperiments, Ab1 inhibited GM-CSF activity more effectively than Ab2when comparing IC50 (Table 5).

TABLE 5 Comparison of IC50 for inhibition of GM-CSF induced IL-8expression. Data from three independent experiments shown in FIG. 5 andmean IC₅₀ are expressed in ng/ml and nM. Experiment Ab2 (ng/ml) Ab2 (nM)Ab1 (ng/ml) Ab1 (nM) A 363 2.4 31.3 0.21 B 514 3.4 92.5 0.62 C 343 2.220.7 0.14 Mean 407 2.7 48.2 0.32Summary

The Humaneered Ab1 bound to GM-CSF with a calculated equilibrium bindingconstant (KD) of 25 pM. Ab2 bound to GM-CSF with a KD of 30.5 pM. Ab2showed a two-fold higher association constant (k_(a)) than Ab1 forGM-CSF while Ab1 showed three-fold slower dissociation kinetics (k_(d))than Ab2. Ab2 and Ab1 showed similar binding activity for glycosylatedand non-glycosylated GM-CSF in an antigen-binding ELISA. A competitionELISA confirmed that both antibodies competed for the same epitope; Ab1showed higher competitive binding activity than Ab2. In addition, Ab1showed higher GM-CSF neutralization activity than Ab2 in aGM-CSF-induced IL-8 induction assay.

Example 3—Administration of a Neutralizing Anti-GM-CSF Antibody in aMouse Model of Immunotherapy-Related Toxicity

A mouse model of immunotherapy-related toxicity can be used to show theefficacy of an anti-GM-CSF antibody for preventing and treatingimmunotherapy-related toxicity. In one model of immunotherapy-relatedtoxicity, mice are injected with CAR T-cells in doses provokingtoxicity. For example, van der Stegen et al. (J. Immunol 191:4589-4598(2013)), incorporated herein by reference, describe a CRS model inducedby the i.p. injection of a single dose of 30×10⁶ cells termed T4⁺ Tcells. T4⁺ T cells are engineered T cells expressing the chimeric Agreceptor (CAR) T1E28z. T cells engineered to express T1E28z areactivated by cells expressing ErbB1- and ErbB4-based dimers and ErbB2/3heterodimer.

To evaluate the efficacy of anti-GM-CSF antibodies for preventing andtreating CRS, mice will be divided in groups (n=10), each groupreceiving either: a) a single i.p. saline injection; b) an i.p.injection of 30×10⁶ T4⁺ T cells; c) an i.p. injection of 30×10⁶ T4⁺ Tcells and 0.25 mg intravenous (i.v.) anti-GM-CSF monoclonal antibody22E9 (a recombinant rat anti-mouse-GM-CSF antibody) co-administered withT4⁺ T cells; d) an i.p. injection of 30×10⁶ T4⁺ T cells and 0.25 mgintranasal (i.n.) anti-GM-CSF antibody 22E9 co-administered with T4⁺ Tcells; e) an i.p. injection of 30×10⁶ T4⁺ T cells and 0.25 mg i.v.anti-GM-CSF antibody 22E9 6 hours before T4⁺ T cells administration; f)an i.p. injection of 30×10⁶ T4⁺ T cells and 0.25 mg i.n. anti-GM-CSFantibody 22E9 6 hours before T4⁺ T cells administration; g) an i.p.injection of 30×10⁶ T4⁺ T cells and 0.25 mg i.v. anti-GM-CSF antibody22E9 2 hours after T4⁺ T cells administration; or h) an i.p. injectionof 30×10⁶ T4⁺ T cells and 0.25 mg i.n. anti-GM-CSF antibody 22E9 2 hoursafter T4⁺ T cells administration. Further doses, administration times,and administration routes will be evaluated.

In order to assess anti-GM-CSF antibody 22E9 effect, organs will becollected from mice, formalin fixed, and subjected to histopathologicanalysis. Blood will be collected and concentrations of human IFNγ,human IL-2, and mouse IL-6, IL-2, IL-4, IL-6, IL-10, IL-17, IFNγ, andTNFα will be assessed by well methods described in the literature, suchas ELISA assay. Mice weight, behavior, and clinical manifestations willbe observed.

Example 4—Anti-GM-CSF Antibody Effect on Immunotherapy

A mouse model can be used to show that GM-CSF antagonists do notnegatively affect the efficacy of cancer immunotherapy. SCID beige micecan be inoculated with a cancer cell line and treated with animmunotherapeutic agent known to induce CRS, as T4⁺ T cells, with orwithout an anti-GM-CSF antibody.

To evaluate whether anti-GM-CSF antibodies affect the efficacy ofimmunotherapy, mice will be divided in groups (n=10), each groupreceiving either: a) a subcutaneous (s.c.) injection of 30×10⁶ SKOV3cells; b) a s.c. injection of 30×10⁶ SKOV3 cells and an i.p. injectionof 30×10⁶ T4⁺ T cells; or c) a s.c. implant of 30×10⁶ SKOV3 cells, ani.p. injection of 30×10⁶ T4⁺ T cells, and an i.v. injection of 0.25 mgof anti-GM-CSF antibody 22E9.

In order to assess anti-GM-CSF antibody 22E9 effect on T4⁺ T cellsefficacy, tumor size will be measured every four days by caliper, andtumor volume calculated by the formula: 0.5×(larger diameter)×(smallerdiameter)². Mice weight, behavior, and clinical manifestations will beobserved. At the end of the experiment, the animals will be sacrificed,and the tumor tissues harvested and weighted.

Example 5—Mouse Model of Human CRS

A mouse model for CRS for investigating the effects of a humanizedanti-GM-CSF monoclonal antibody in treating or preventing CRS wasdeveloped. (FIG. 17a .-17 b.).

Method: The model used is a primary AML model. Immunocompromised NSG-Smice that were additionally transgenic for human SCF, IL-3, and GM-CSFwere engrafted with AML blasts derived from AML patients that were CD123positive. After 2-4 weeks, they were bled to confirm engraftment andachievement of high disease burden. The mice were then treated with highdoses of CAR-T123 at 1×10⁶ cells, which is 10 times higher than dosespreviously studied.

Results: It was observed that within 1-2 weeks after CAR-T cellinjection, these mice developed an illness characterized by weakness,emaciation, hunched bodies, withdrawal, and poor motor response. Themice eventually died of their disease within 7-10 days. The symptomscorrelate with massive T-cell expansion in the mice and with elevationof multiple human cytokines, such as IL-6, MIP 1α, IFN-γ, TNFα, GM-CSF,MIP1β, and IL-2, and in a pattern that resembles what is seen in humanCRS after CAR-T cell therapy. GM-CSF fold change was significantlygreater than other cytokines. (FIG. 17 a-b).

Example 6—Generation of GM-CSF Knockout CAR-Ts

GM-CSF CRISPR knockout T cells were generated and shown to exhibitreduced expression of GM-CSF but similar levels of other cytokines anddegranulation, which showed immune cell functionality. (See FIGS.15a-15g ).

Example 7—Anti-GM-CSF Neutralizing Antibody Does Not Inhibit CAR-TMediated Killing, Proliferation, or Cytokine Production but NeutralizesGM-CSF

Anti-GM-CSF neutralizing antibody does not inhibit CAR-T mediatedkilling, proliferation, or cytokine production but successfullyneutralizes GM-CSF. (See FIGS. 16a-16i ).

Example 8—Anti-GM-CSF Neutralizing Antibody Does Not Inhibit CAR-TEfficacy In Vivo

Humanized anti-GM-CSF monoclonal antibody, a neutralizing hGM-CSFantibody, does not inhibit CAR-T efficacy in vivo (FIG. 18a-18c ). CAR-Tefficacy in a xenograft model in combination with an anti-GM-CSFneutralizing antibody in accordance with embodiments described herein.As shown in FIG. 18a , NSG mice were injected with NALM-6-GFP/Luciferasecells (human, peripheral blood leukemia pre-B cell), and bioluminescentimaging (BLI0 was performed to confirm tumor growth. Mice were treatedwith either (1) anti-GM-CSF antibody (10 mg/Kg daily for ten days) and(a) CART19 or (b) untransduced human T cells (UTD) 1×10⁶ cells or (2)IgG control antibody (10 mg/Kg daily for ten days) and (a) CART19 or (b)untransduced human T cells (UTD) 1×10⁶ cells. FIGS. 18b and 18cdemonstrate that the anti-GM-CSF neutralizing antibody did not inhibitCAR-T efficacy in vivo.

Example 9—Anti-GM-CSF Neutralizing Antibody Does Not Impair CAR-T Impacton Survival

In vitro and in vivo preclinical data show anti-GM-CSF neutralizingantibody (a humanized anti-GM-CSF monoclonal antibody) does not impairCAR-T impact on survival in mouse models. (FIG. 19).

The anti-GM-CSF neutralizing antibody does not impede CAR-T cellfunction in vivo in the absence of PBMCs. Survival shown to be similarfor CAR-T+control and CAR-T+anti-GM-CSF neutralizing antibody.

Example 10—Anti-GM-CSF Neutralizing Antibody May Increase CAR-TExpansion

In vitro and In vivo preclinical data show anti-GM-CSF neutralizingantibody (a humanized anti-GM-CSF monoclonal antibody) may increaseCAR-T Expansion (FIG. 20). The anti-GM-CSF neutralizing antibody mayincrease in vitro CAR-T cancer cell killing. The antibody increasesproliferation of CAR-T cells and could improve efficacy. CAR-Tproliferation increased by the GM-CSF neutralizing antibody in presenceof PBMCs. (It was not affected without PBMCs). The antibody did notinhibit degranulation, intracellular GM-CSF production, or IL-2production.

Example 11—CAR-T Expansion Associated with Improved Overall ResponseRate

CAR-T expansion associated with improved overall response rate. (FIG.21). CAR AUC (area under the curve) defined as cumulative levels ofCAR+cells/μL of blood over the first 28 days post CAR-T administration.P values calculated by Wilcoxon rank sum test. (Neelapu, et al ICML 2017Abstract 8).

Example 12—Study Protocol for an Anti-GM-CSF Neutralizing Antibody inAccordance with Embodiments Described Herein

Study protocol for an anti-GM-CSF neutralizing antibody (a humaneeredanti-GM-CSF monoclonal antibody) in accordance with embodimentsdescribed herein. (See FIG. 22). CRS and NT to be assessed daily whilehospitalized and at clinic visit for first 30 days. Eligible subjects toreceive GM-CSF neutralizing antibody on days −1, +1, and +3 of CAR-Ttreatment. Tumor assessment to be performed at baseline and months 1, 3,6, 9, 12, 18, and 24. Blood samples (PBMC and serum) days −5, −1, 0, 1,3, 5, 7, 9, 11, 13, 21, 28, 90, 180, 270, and 360.

Example 13—GM-CSF Depletion Increases CAR-T Cell Expansion

GM-CSF depletion increases CAR-T cell expansion. (FIG. 23A-23B) FIG. 23Ashows increased ex-vivo expansion of GM-CSF^(k/o) CAR-T cells comparedto control CAR-T cells. FIG. 23b demonstrates more robust proliferationafter in vivo treatment with an anti-GM-CSF neutralizing antibody (ahumaneered anti-GM-CSF monoclonal antibody) in accordance withembodiments described herein.

Example 14—Safety Profile of an Anti-GM-CSF Neutralizing Ab in >100Human Patients*

Phase I:

Single-dose, dose escalation in healthy adult volunteers. Objectiveswere to analyze Safety/tolerability, PK, and Immunogenicity.

Enrollment/dose:

(n=12)

3/1 mg/kg

3/3 mg/kg

3/10 mg/kg

3/placebo

Safety Results:

-   -   Clean Safety Profile:    -   No drug related serious adverse effects (SAE)    -   Non-immunogenic        Phase II:

1) Dose at weeks 0, 2, 4, 8, 12 in rheumatoid arthritis patients.Objectives were to analyze Efficacy, Safety/tolerability, PK, andImmunogenicity.

Enrollment/dose:

(n=9)

7/600 mg

2/placebo

Safety Results:

-   -   Clean Safety Profile:    -   No drug related serious adverse effects (SAE)    -   Non-immunogenic

2) Dose at weeks 0, 2, 4, 8, 12, 16 20 in severe asthma patients.Objectives were to analyze Efficacy, Safety/tolerability, PK, andImmunogenicity.

Enrollment/dose:

(n=160)

78/400 mg

82/placebo

Safety Results:

-   -   Clean Safety Profile:    -   No drug related serious adverse effects (SAE)    -   Non-immunogenic        * 94 patients in studies depicted above, plus 12 patients in        ongoing CMML Phase I trial, where drug is well tolerated; an        additional 76 patients received a chimeric version of a GM-CSF        neutralizing Ab (KB002) and showed a similar safety profile.

All studies randomized double-blind placebo-controlled, IVadministration. (See FIG. 24.)

Example 15—Effect of Anti-GM-CSF Antibody on CART Activity and Toxicity

The study will investigate the effect of GMCSF blockade with anti-GM-CSFantibody on chimeric antigen receptor T cells (CART) activity andtoxicity. This can be accomplished through these two AIMS:

AIM #1: to investigate the effect of GMCSF blockade with anti-GM-CSFantibody on CART cell effector functions

AIM #2: To study the effect of GMCSF blockade with anti-GM-CSF antibodyon reducing cytokine release syndrome after CART cell therapy Researchstrategy. The following experiments are proposed:

In vitro studies of the combination of four different doses of GMCSFblockade with anti-GM-CSF antibody with CART cells (cytokine production(30 plex Lumiex, including GM-CSF, IL-2, INFg, IL-6, IL-8, MCP-1),antigen specific killing, degranulation, proliferation and exhaustion),in the presence or absence of myeloid cells using the model: CART19against ALL.

In vivo studies of the combination of different doses of GMCSF blockadewith anti-GM-CSF antibody (with and without murine GMCSF blockade) withCART cells, using two models:

CD19 positive cell line (NALM6) engrafted xenografts, treated withCART19 with or without anti-GM-CSF antibody; and

Patient derived xenografts with primary ALL, and then treated withCART19 with or without anti-GM-CSF antibody.

Mice will be dosed i.p with anti-GM-CSF antibody 10 mg/kg immediatelyprior to CART cell implantation and 10 mg/kg/day for 10 days. Mice willbe followed for tumor response and survival. Retro-orbital bleedingswill be obtained starting one week after CART cell therapy and weeklyafterwards. Disease burden, T cell expansion kinetics, expression ofexhaustion markers and cytokine levels (30 Plex) will be analyzed. Atthe completion of the experiment, spleens and bone marrows will beharvested and analyzed for tumor characteristics and CAR-T cell numbers.

In vivo studies of the combination of GMCSF blockade with anti-GM-CSFantibody (with or without murine GMCSF blockade) with CART cells in CRSmodels (in this model, high doses of CART cells will be used to elicitCRS), in the presence of PBMCs, using the following model:

Primary ALL patient derived xenografts, then treated with CART19 with orwithout anti-GM-CSF antibody.

Mice will be dosed i.p. with anti-GM-CSF antibody 10 mg/kg immediatelyprior to CART cell implantation and 10 mg/kg/day for 10 days. Mice willbe followed for tumor response, CRS toxicity symptoms and survival.Retro-orbital bleedings will be obtained at baseline, 2 days post, oneweek-post CART cell therapy and weekly afterwards. Disease burden, Tcell expansion kinetics, expression of exhaustion markers and cytokinelevels (30 Plex) will be analyzed. At the completion of the experiment,spleens and bone marrows will be harvested and analyzed for tumorcharacteristics and CAR-T cell numbers

In Vivo Neurotoxicity Assays

Using models discussed in #3 above, mice will be imaged with MRI whilesick to assess for development of neurotoxicity after CART cell therapy.Images will be compared between mice that received CART cells andanti-GM-CSF antibody vs control antibody. Repeat experiments will beperformed. Mice will be euthanized 14 days after CART cells in theserepeat experiments. Brain tissue will be analyzed for cytokines withmultiplex assays, for the presence of monocytes, human T cells, and forintegrity of blood brain barrier by IHC, flow and microscopy.

Example 16 Anti-hGM-CSF Neutralizing Antibody Reduces Neuroinflammationin CAR-T Cell Related Neurotoxicity (NT)

There is extensive scientific rationale implicating GM-CSF as essentialto the initiation of cytokine release syndrome (CRS), neurotoxicity (NT)and the inflammatory cascade seen following initiation of CAR-T celltherapy. The hypothesis studied is that blocking soluble GM-CSF with theneutralizing antibody (lenzilumab) will abrogate or prevent the onsetand severity of both CRS and NT observed with CAR-T cell therapy.Importantly, CAR-T cell activity should be preserved or improved ifpossible. The experimental design tests the effects of GM-CSF blockadewith anti-GM-CSF antibody (lenzilumab) on CAR-T cell effector functions,CAR-T efficacy in a tumor xenograft model, development of CRS in a CRSxenograft model and the development of NT using MRI imaging andvolumetric analysis to quantify the neuro-inflammation seen with CAR-Tcell therapy. In vitro and in vivo experiments with CAR-T+/−lenzilumabboth in the presence and absence of human PBMCs were studied. (seeExamples 9 and 10, FIGS. 19 and 20 a-20 b).

Methods

In vitro studies were conducted to evaluate the combination of GM-CSFneutralizing antibody lenzilumab with human CD19+ CAR-T cells onantigen-specific killing, degranulation, proliferation and exhaustion inthe presence or absence of human PBMCs.

To assess the impact of anti-GM-CSF antibody (lenzilumab) on CAR-T cellproliferation and efficacy, in vivo studies were subsequently conductedusing the following model (with and without murine GM-CSF blockade):

Effector/Target Control Experiments:

CD19 positive cell line (NALM6) engrafted xenografts, treated withCART19 with or without anti-GM-CSF antibody (lenzilumab) in the absenceof human PBMCs.

NSG mice were dosed i.p. with anti-GM-CSF antibody (lenzilumab) 10 mg/kgimmediately prior to CAR-T cell implantation and at the same dose everyday thereafter for 10 days and followed to assess tumor response andsurvival. Retro-orbital bleedings were obtained starting one week afterCAR-T cell therapy and weekly afterwards. Disease burden, T cellexpansion kinetics, expression of exhaustion markers and cytokine levels(30 Plex) were also analyzed. At the completion of the experiment,spleens and bone marrows were harvested and analyzed for tumorcharacteristics and CAR-T cell numbers.

CRS/NT Experiments: Patient Derived Xenografts with Primary ALL,Subsequently Treated with CART19 with or without Lenzilumab in thePresence of Human PBMCs:

To assess the impact of lenzilumab on abrogating or preventing the onsetand severity of CAR-T induced CRS and NT, in vivo studies were conductedwith human CAR-T cells (with and without murine GM-CSF blockade) in aCRS model (where high doses of CAR-T cells were used to illicit CRS) inthe presence of PBMCs using primary ALL patient derived xenografts,treated with CART19 with and without lenzilumab. NSG mice were dosedi.p. with lenzilumab 10 mg/kg immediately prior to CAR-T cellimplantation and every day thereafter for 10 days. Mice were followedfor tumor response, survival, CRS and NT symptoms. Brain MRI scans weretaken at baseline, during and at the end of CAR-T cell therapy andvolumetric analysis was conducted to assess and quantifyneuro-inflammation and MRI T1 hyperintensity across treatment arms. Bodyweight and retro-orbital bleedings were obtained at baseline, 2 dayspost, one week-post CAR-T cell therapy and weekly afterwards. Diseaseburden, T cell expansion kinetics, expression of exhaustion markers andcytokine levels (30 Plex) were analyzed. At the completion of theexperiment, spleens and bone marrows were harvested and analyzed fortumor characteristics and CAR-T cell numbers.

Results

In Vitro Model

In this experiment, the impact of GM-CSF neutralization with lenzilumabon CAR-T cell effector functions was investigated. It was demonstratedthat GM-CSF is secreted by CAR-T cells at very high levels (over 1,500pg/ml) and the use of lenzilumab completely neutralized GM-CSF but didnot inhibit CAR-T degranulation, intracellular GM-CSF production or IL2production. Moreover, lenzilumab did not inhibit CAR-T antigen specificproliferation or CAR-T killing. Effector-to-target rations (E:T) weresimilar with CAR-T+lenzilumab vs. CAR-T+control antibody, p=ns (FIGS.16a-16d and 16j ).

In vivo Models:

Effector/Target Control Experiments:

To study the effect of lenzilumab on CART19 cell function in vivo, weengrafted immuno-compromised NOD-SCID-g−/− with the CD19+ ALL cell lineNALM6 in the absence of human PBMCs. Treatment with CART19 combined withlenzilumab resulted in potent anti-tumor activity and improved overallsurvival, similar to CART19 with control antibody despite completeneutralization of GM-CSF levels in these mice, indicating that GM-CSFdoes not impair CAR-T cell activity in vivo in the absence of PBMCs(FIGS. 16f and 16g ).

CRS and NT Experiments:

Using human ALL blasts, human CD19 CAR-T, and human PBMCs, lenzilumab incombination with CAR-T cell therapy was found to reduceneuro-inflammation by ˜90% compared to CAR-T alone as assessed byquantitative MRI T1 hyperintensity. This is a landmark finding and thefirst time it has been demonstrated in vivo that the neuroinflammationcaused by CAR-T cell therapy can be effectively abrogated. MRI imagesfollowing lenzilumab plus CAR-T cell therapy were similar to baselinepre-treatment scans, in sharp contrast to MRI images following controlantibody plus CAR-T cell therapy which showed marked increasedinflammation. Moreover, a decrease in myeloid cells was seen in thebrains of mice treated with lenzilumab plus CAR-T compared to micetreated with CAR-T and control antibody. This finding is consistent withdata reported in clinical trials with CD19 CAR-T cell therapy where anincrease in myeloid cells was observed in the CSF of patients withsevere grade >3 neurotoxicity. In addition, lenzilumab in combinationwith CAR-T cell therapy was found to reduce the onset and severity ofCRS as compared to CAR-T plus control antibody. This finding issupported by the statistically significant reduction in body weight seenin mice treated with CAR-T plus control, the most objective marker andhallmark symptom of CRS seen in vivo. In mice treated with lenzilumabplus CAR-T, body weight was maintained at baseline levels as compared toCAR-T plus control (p<0.05). Moreover, mice treated with CAR-T pluscontrol antibody displayed physical symptoms consistent with CRSincluding hunched posture, withdrawal, and weakness while mice treatedwith CAR-T plus lenzilumab appeared healthy. Importantly, lenzilumabplus CAR-T also demonstrates a significant 5-fold increase in theproliferation of CAR-T cells compared to CAR-T plus control in theseCRS/NT experiments that included PBMCs. It has been previously shown inclinical trials with various CD19 CAR-T cell therapies that improvedCAR-T proliferation or expansion correlates with improved efficacy(including ORR, CR), suggesting that lenzilumab may potentially improveanti-tumor response. This finding may be in part explained by a decreasein MDSC expansion and trafficking which is known to be promulgated byGM-CSF. Lastly, the combination of lenzilumab plus CAR-T results insignificantly better leukemic control as quantified by flow cytometrycompared to CAR-T and control antibody. Compared to untreated mice(which had 500,000 to 1.5M leukemic cells) and CAR-T plus controlantibody (which had between 15,000 and 100,000 leukemic cells),treatment with CAR-T plus lenzilumab led to a significant reduction inthe number of leukemic cells (decreased to between 500 and 5,000 cells)with improved overall disease control (see FIGS. 25A-25D).

The MRI images in FIG. 25A shows a clear improvement in neurotoxicity(NT) (neuroinflammation) in the brains of mice administered CAR-T cellsand anti-GM-CSF neutralizing antibody in accordance with embodimentsdescribed herein. In contrast, the brains of mice administered CAR-Tcells and a control antibody showed signs of neurotoxicity in the MRIimages. FIG. 25B graphically illustrates that the NT was reduced by 90%in the mice of Group 1 compared to the NT increased in Group 2 mice. Theextent of quantitative improvement (90% reduction in NT) afteradministration of CAR-T cells and anti-GM-CSF neutralizing antibody inaccordance with embodiments described herein was an unexpected finding.

Conclusions

Anti-GM-CSF antibody (Lenzilumab), when combined with CAR-T cell therapydemonstrates the potential to prevent the onset and severity of CRS andNT, while improving CAR-T expansion/proliferation and overall leukemiccontrol in-vivo using human ALL blasts, human CD19 CAR-T and humanPBMCs. This is the first time it has been demonstrated that CAR-Tinduced neurotoxicity can be abrogated in-vivo. Pivotal clinical trialswith lenzilumab in combination with CAR-T cell therapy are planned tovalidate these findings of improved safety and efficacy.

Example 17 GM-CSF Blockade During Chimeric Antigen Receptor T CellTherapy Reduces Cytokine Release Syndrome and Neurotoxicity and MayEnhance Their Effector Functions

Despite its efficacy, chimeric antigen receptor T-cell therapy (CART) islimited by the development of cytokine release syndrome (CRS) andneurotoxicity (NT). While CRS is related to extreme elevation ofcytokines and massive T cell expansion, the exact mechanisms for NT havenot yet been elucidated. Preliminary studies suggest that NT might bemediated by myeloid cells that cross the blood brain barrier. This issupported by correlative analysis from CART19 pivotal trials where CD14+cell numbers were increased in the cerebrospinal fluid of patients thatdeveloped severe NT (Locke et al, ASH 2017). Therefore, the aimed ofthis study was to investigate the role of GM-CSF neutralization inpreventing CRS and NT after CART cell therapy via monocyte control.

First, the effect of GM-CSF blockade on CART cell effector functions wasinvestigated. Here, the human GM-CSF neutralizing antibody (lenzilumab,Humanigen, Burlingame, Calif.) was used that has been shown to be safein phase II clinical trials. Lenzilumab (10 ug/kg) neutralizes GM-CSFwhen CART19 cells are stimulated with the CD19+ Luciferase+ acutelymphoblastic leukemia (ALL) cell line NALM6, but does not impair CARTcell function in vitro. It was found that malignancy associatedmacrophages reduce CART proliferation. GM-CSF neutralization withlenzilumab results in enhanced CART cell antigen specific proliferationin the presence of monocytes. To confirm this in vivo, NOD-SCID-g−/−mice were engrafted with high disease burdens of NALM6 and treated withlow doses of CART19 or control T cells (to induce tumor relapse), incombination with lenzilumab or isotype control antibody. The combinationof CART19 and lenzilumab resulted in significant anti-tumor activity andoverall survival benefit compared to control T cells (FIG. 26A), similarto mice treated with CART19 combined with isotype control antibody,indicating that GM-CSF neutralization does not impair CART cell activityin vivo. This anti-tumor activity was validated in an ALL patientderived xenograft model.

Next, explored was the impact of GM-CSF neutralization on CART cellrelated toxicities in a novel patient derived xenograft model. Here,NOD-SCID-g−/− mice were engrafted with leukemic blasts (1-3×106 cells)derived from patients with high risk relapsed ALL. Mice were thentreated with high doses of CART19 cells (2-5×106 intravenously). Fivedays after CART19 treatment, mice began to develop progressive motorweakness, hunched bodies, and weight loss that correlated with massiveelevation of circulating human cytokine levels. Magnetic ResonanceImaging (MRI) of the brain during this syndrome showed diffuseenhancement and edema, associated with central nervous system (CNS)infiltration of CART cells and murine activated myeloid cells. This issimilar to what has been reported in CART19 clinical trials in patientswith severe NT. The combination of CART19, lenzilumab (to neutralizehuman GM-CS) and murine GM-CSF blocking antibody (to neutralize mouseGM-CSF) resulted in prevention of weight loss (FIG. 26B), decrease incritical myeloid cytokines (FIGS. 26C-26D), reduction of cerebral edema(FIG. 26E), enhanced leukemic disease control in the brain (FIG. 26F),and reduction in brain macrophages (FIG. 26G).

Finally, it was hypothesized that disrupting GM-CSF through CRISPR/Cas9gene editing during the process of CART cell manufacture would result infunctional CART cells with reduced secretion of GM-CSF. Guide RNAtargeting exon 3 of the GM-CSF gene was designed and GM-CSF^(k/o) CART19cells were generated. The preliminary data suggest that these CARTsproduce significantly less GM-CSF upon activation but continue toexhibit similar production of other cytokines and exhibit normaleffector functions in vitro (FIG. 26H). Using the NALM6 high tumorburden relapse xenograft model as described above, GM-CSF^(k/o) CART19cells resulted in slightly enhanced disease control compared to CART19cells (FIG. 26I).

Thus, modulating myeloid cell behavior through GM-CSF blockade can helpcontrol CART mediated toxicities and may reduce their immunosuppressivefeatures to improve leukemic control. These studies illuminate a novelapproach to abrogate NT and CRS through GM-CSF neutralization that alsopotentially enhances CART cell functions. Based on these results, aphase II clinical trial has been designed using lenzilumab as a modalityto prevent CART related toxicities in patients with diffuse large B celllymphoma.

Example 18 GM-CSF Neutralization In Vitro Enhances CAR-T CellProliferation in the Presence of Monocytes and does not Impair CAR-TCell Effector Function

Cells Lines and Primary Cells

The NALM6 cell line was purchased from ATCC, Manassas, Va., USA, and theMOLM13 cell line was a gift from the Jelinek Laboratory at the MayoClinic (purchased from DSMZ, Braunschweig, Germany). These cell lineswere transduced with a luciferase-ZsGreen lentivirus (Addgene,Cambridge, Mass., USA) and sorted to 100% purity. Cell lines werecultured in R10 (made with RPMI 1640 (Gibco, Gaithersburg, Md., US), 10%Fetal Bovine Serum (FBS, Millipore Sigma, Ontaria, Canada), and 1%Penicillin-Streptomycin-Glutamine (Gibco, Gaithersburg, Md., US).Primary cells were obtained from the Mayo Clinic Biobank for patientswith acute leukemia under a Mayo Clinic Institutional Review Board (IRB)approved protocol. The use of recombinant DNA in the laboratory wasapproved by the Mayo Clinic Institutional Biosafety Committee (IBC).

Primary Cells and CAR-T Cells

Peripheral blood mononuclear cells (PBMC) were isolated fromde-identified normal donor blood apheresis cones obtained under a MayoClinic IRB approved protocol, using SepMate tubes (STEMCELLTechnologies, Vancouver, Canada). T cells were separated with negativeselection magnetic beads using EasySep™ Human T Cell Isolation Kit(STEMCELL Technologies, Vancouver, Canada). Monocytes were isolatedusing a Human Monocyte Isolation Kit from Miltenyi Biotec, BergischGladbach, Germany, which isolates CD14+ monocytes. Primary cells werecultured in T Cell Medium made with X-Vivo 15 (Lonza, Walkersville, Md.,USA) supplemented with 10% human serum albumin (Corning, N.Y., USA) and1% Penicillin-Streptomycin-Glutamine (Gibco, Gaithersburg, Md., USA).CART19 cells were generated through the lentiviral transduction ofnormal donor T cells as described herein below. Second generation CART19constructs were de novo synthesized (IDT) and cloned into athird-generation lentivirus under the control of the EF-1α promotor. TheCD19 directed single chain variable region fragment was derived from theclone FMC63. A second generation 4-1BB co-stimulated (FMC63-41BBz) CARconstruct was synthesized and used for these experiments. Lentiviralparticles were generated through the transient transfection of plasmidinto 293T virus producing cells (gift from the Ikeda lab, Mayo Clinic),in the presence of Lipofectamine 3000 (Invitrogen, Carlsbad, Calif.,USA), VSV-G and packaging plasmids (Addgene, Cambridge, Mass., USA). Tcells isolated from normal donors were stimulated using Cell TherapySystems Dynabeads CD3/CD28 (Life Technologies, Oslo, Norway) at a 1:3ratio and then transduced with lentivirus particles 24 hours afterstimulation at a multiplicity of infection (MOI) of 3.0. To determinetiters and subsequently MOI, after lentivirus particles wereconcentrated, titers were determined by transducing 1×10⁵ primary Tcells in 100 ul of T cell medium with 50 ul of lentivirus. First, Tcells were stimulated with CD3/CD28 beads and then transduced withlentivirus particles 24 hours later. Transduction was performed intriplicates and at serial dilutions. Fresh T cell medium was added oneday later. Two days later, cells were harvested, washed twice with PBS,and CAR expression on T cells was determined by flow cytometry. Titerswere determined based on the percentage of CAR positive cells(percentage of CAR+ cells×T cell count at transduction×the specificdilution/volume) and expressed as transducing units/mL (TU/mL). Magneticbead removal was performed on Day 6 and CAR-T cells were harvested andcryopreserved on Day 8 for future experiments. CAR-T cells were thawedand rested in T cell medium 12 hours prior to their use in experiments.

GM-CSF Neutralizing Antibody and Isotype Controls

Lenzilumab (Humanigen, Burlingame, Calif.), an hGM-CSF neutralizingantibody in accordance with embodiments described herein and asdescribed in U.S. Pat. Nos. 8,168,183 and 9,017,674, each of which isincorporated herein by reference in its entirety, is a novel, first inclass Humaneered® monoclonal antibody that neutralizes human GM-CSF. Forin vitro experiments, lenzilumab or InVivoMAb human IgG1 isotype control(BioXCell, West Lebanon, N.H., USA), 10 ug/mL was used. For in vivoexperiments, 10 mg/kg of lenzilumab or isotype control wereintraperitoneally injected daily for 10 days beginning on the day ofCART19 injection. In some experiments, anti-mouse GM-CSF neutralizingantibody (InVivoMAb anti-mouse GM-CSF, BioXCel, West Lebanon, N.H., USA)or the corresponding isotype control (InVivoMAb rat IgG2a isotypecontrol BioXCel, West Lebanon, N.H., USA) was also used, as indicated inthe experimental schema.

T Cell Functional Experiments

Cytokine assays were performed 24 or 72 hours after a co-culture ofCAR-T cells with their targets at a 1:1 ratio as indicated. Human HighSensitivity T Cell Magnetic Bead Panel (Millipore Sigma, Ontario,Canada), Milliplex Human Cytokine/Chemokine MAGNETIC BEAD Premixed 38Plex Kit (Millipore Sigma, Ontario, Canada), or Milliplex MouseCytokine/Chemokine MAGNETIC BEAD Premixed 32 Plex Kit (Millipore Sigma,Ontario, Canada) were performed on supernatants collected from theseexperiments or serum, as indicated. This was analyzed using Luminex(Millipore Sigma, Ontario, Canada). Intracellular cytokine analysis andT cell degranulation assays were performed following incubation of CAR-Tcells with targets at a 1:5 ratio for 4 hours, in the presence ofmonensin (BioLegend, San Diego, Calif., USA), hCD49d (BD Biosciences,San Diego, Calif., USA), and hCD28 (BD Biosciences, San Diego, Calif.,USA). After 4 hours, cells were harvested and intracellular staining wasperformed after surface staining, followed by fixation andpermeabilization with fixation medium A and B (Life Technologies, Oslo,Norway). For proliferation assays, CFSE (Life Technologies, Oslo,Norway) labeled effector cells (CART19), and irradiated target cellswere co cultured at a 1:1 ratio. In some experiments, CD14+ monocyteswere added to the co-culture at a 1:1:1 ratio as indicated. Cells wereco-cultured for 3-5 days, as indicated in the specific experiment andthen cells were harvested and surface staining with anti-hCD3(eBioscience, San Diego, Calif., USA) and LIVE/DEAD™ Fixable Aqua DeadCell Stain Kit (Invitrogen, Carlsbad, Calif., USA) was performed.PMA/ionomycin (Millipore Sigma, Ontario, Canada) was used as a positivenon-specific stimulant of T cells, at different concentrations asindicated in the specific experiments. For killing assays, theCD19⁺Luciferase⁺ ALL cell line NALM6 or the CD19⁻Luciferase⁺ controlMOLM13 cells were incubated at the indicated ratios with effector Tcells for 24, 48, or 72 hours as listed in the specific experiment.Killing was calculated by bioluminescence imaging on a Xenogen IVIS-200Spectrum camera (PerkinElmer, Hopkinton, Mass., USA) as a measure ofresidual live cells. Samples were treated with 1 ul D-luciferin (30ug/mL) per 100 ul sample volume (Gold Biotechnology, St. Louis, Mo.,USA), for 10 minutes prior to imaging.

Multi-Parametric Flow Cytometry

Anti-human and anti-mouse antibodies were purchased from Biolegend,eBioscience, or BD Biosciences (San Diego, Calif., USA). Cells wereisolated from in vitro culture or from peripheral blood of animals.After BD FACS lyse (BD Biosciences, San Diego, Calif., USA), they werewashed twice in phosphate-buffered saline supplemented with 2% FBS(Millipore Sigma, Ontario, Canada) and 1% sodium azide (Ricca Chemical,Arlington, Tex., USA) and stained at 4° C. For cell number quantitation,Countbright beads (Invitrogen, Carlsbad, Calif., USA) were usedaccording to the manufacturer's instructions (Invitrogen, Carlsbad,Calif., USA). In all analyses, the population of interest was gatedbased on forward vs side scatter characteristics, followed by singletgating, and live cells were gated following staining with LIVE/DEAD™Fixable Aqua Dead Cell Stain Kit (Invitrogen, Carlsbad, Calif., USA).Surface expression of CAR was detected by staining with a goatanti-mouse F(ab′)2 antibody (Invitrogen, Carlsbad, Calif., USA). Flowcytometry was performed on three-laser cytometers, Canto II (BDBiosciences, San Diego, Calif., USA) and CytoFLEX (Beckman Coulter,Chaska, Minn., USA). Analyses were performed using FlowJo X10.0.7r2software (Ashland, Oreg., USA) and Kaluza 2.0 software (Beckman Coulter,Chaska, Minn., USA).

Results

If GM-CSF neutralization after CAR-T cell therapy is to be utilized as astrategy to prevent CRS and neurotoxicity, it must not inhibit CAR-Tcell efficacy. Therefore, the initial experiments aimed to investigatethe impact of GM-CSF neutralization on CAR-T cell effector functions.CART19 cells were co-cultured with or without the CD19⁺ ALL cell lineNALM6 in the presence of lenzilumab (hGM-CSF neutralizing antibody) oran isotype control (IgG). It was established that lenzilumab, but notIgG control antibody, was indeed able to completely neutralize hGM-CSF(FIG. 27A) but did not inhibit CAR-T cell antigen specific proliferation(FIG. 27B). When CART19 cells were co-cultured with the CD19⁺ cell lineNALM6 in the presence of monocytes, lenzilumab in combination withCART19 demonstrated an exponential increase in antigen specific CART19proliferation compared to CART19 plus isotype control IgG (P<0.0001,FIG. 27C). To investigate CAR-T specific cytotoxicity, either CART19 orcontrol UTD T cells were cultured with the luciferase+CD19⁺ NALM6 cellline and treated with either isotype control antibody or GM-CSFneutralizing antibody (FIG. 27D). GM-CSF neutralizing antibody treatmentdid not inhibit the ability of CAR-T cells to kill NALM6 target cells(FIG. 27D). Overall, these results indicate that lenzilumab does notinhibit CAR-T cell function in vitro and enhances CART19 cellproliferation in the presence of monocytes, suggesting that GM-CSFneutralization may improve CAR-T cell mediated efficacy.

Example 19 GM-CSF Neutralization In Vivo Enhances CAR-T Cell Anti-TumorActivity in Xenograft Models

Xenograft Mouse Models

Male and female 8-12 week old NOD-SCID-IL2rγ^(−/−) (NSG) mice were bredand cared for within the Department of Comparative Medicine at the MayoClinic under a breeding protocol approved by the Mayo ClinicInstitutional Animal Care and Use Committee (IACUC). Mice weremaintained in an animal barrier space that is approved by the IBC forBSL2+ level experiments.

NALM6 Cell Line Xenografts

The CD19⁺, luciferase ALL NALM6 cell line was used to establish ALLxenografts, under an IACUC approved protocol. Here, 1×10⁶ cells wereinjected intravenously (IV) via a tail vein injection. 4-6 days afterinjection, mice underwent bioluminescent imaging using a XenogenIVIS-200 Spectrum camera (PerkinElmer, Hopkinton, Mass., USA), toconfirm engraftment. Imaging was performed 10 minutes after theintraperitoneal (IP) injection of 10 ul/g D-luciferin (15 mg/mL, GoldBiotechnology, St. Louis, Mo., USA). Mice were then randomized based ontheir bioluminescent imaging to receive different treatments as outlinedin the specific experiments. Typically, 1-1.5×10⁶ CAR-T cells (and anequivalent of total T cell number of untransduced (UTD) T cells) wereinjected IV per mouse. Transduction efficiency of CAR-T cells wastypically approximately 50%. For example, with a 50% transductionefficiency of CAR-T cells, mice that received 1.5×10⁶ CAR-T cellsreceived 3 million total T cells, and the corresponding UTD micereceived 3×10⁶ UTD. Weekly imaging was performed to assess and followdisease burden. Bioluminescent images were acquired using a XenogenIVIS-200 Spectrum camera (PerkinElmer, Hopkinton, Mass., USA) andanalyzed using Living Image version 4.4 (Caliper LifeSciences,PerkinElmer). Tail vein bleeding was done 7-8 days after injection ofCAR-T cells to assess T cell expansion and cytokines and chemokines, andsubsequently as needed. Mouse peripheral blood was subjected to redblood cell lysis using BD FACS Lyse (BD Biosciences, San Diego, Calif.,USA) and then used for flow cytometric studies. Antibody treated micecommenced daily antibody therapy (10 mg/kg lenzilumab or isotypecontrol) IP on the same day of CART cell therapy for a total of 10 days.

RNA-Seq on Mouse Brain Tissue

RNA was isolated using miRNeasy Micro kit (Qiagen, Gaithersburg, Md.,USA) and treated with RNase-Free DNase Set (Qiagen, Gaithersburg, Md.,USA). RNA-seq was performed on an Illumina HTSeq 4000 (Illumina, SanDiego, Calif., USA) by the Genome Analysis Core at Mayo Clinic. Thebinary base call data was converted to fastq using Illumina bcl2fastqsoftware. The adapter sequences were removed using Trimmomatic, asdescribed by Bolger, A M, et al., Bioinformatics. 2014;30(15):2114-2120. 10.1093/bioinformatics/btu170, which is herebyincorporated by reference in its entirety, and FastQC as described byLeggett R M, et al., Front Genet. 2013; 4:288. Prepublished on 2014 Jan.2 as DOI 10.3389/fgene.2013.00288, which is hereby incorporated byreference in its entirety, was used to check for quality. The latesthuman (GRCh38) and mouse (GRCm38) reference genomes were downloaded fromNCBI. Genome index files were generated using STAR, as described byDobin A, et al., Bioinformatics. 2013; 29(1):15-21.10.1093/bioinformatics/bts635, which is hereby incorporated by referencein its entirety, and the paired end reads were mapped to the genome foreach condition. HTSeq, as described by Anders S, et al., Bioinformatics.2015; 31(2):166-169. Prepublished on 2014 Sep. 28 as DOI10.1093/bioinformatics/btu638, which is hereby incorporated by referencein its entirety, was used to generate expression counts for each gene,and DeSeq2³⁸, as described by Love M I, et al., Genome Biol. 2014;15(12):550. Prepublished on 2014 Dec. 18 as DOI10.1186/s13059-014-0550-8, which is hereby incorporated by reference inits entirety was used to calculate differential expression. Geneontology was assessed using Enrichr, as described by Kuleshov M V etal., Nucleic Acids Research 2016; 44(W1):W90-W97. 10.1093/nar/gkw377,which is hereby incorporated by reference in its entirety. FIG. 35summarizes the steps detailed above. RNA sequencing data are availableat the Gene Expression Omnibus under accession number GSE121591.

Statistics

Prism Graph Pad (La Jolla, Calif., USA) and Microsoft Excel (Microsoft,Redmond, Wash., USA) were used to analyze data. The high cytokineconcentrations in the heat map were normalized to “1” and lowconcentrations normalized to “0” via Prism. Statistical tests aredescribed in the figure legends.

Results

To confirm that GM-CSF depletion does not inhibit CART19 effectorfunctions, the role of GM-CSF neutralization with lenzilumab on CART19antitumor activity was investigated in xenograft models. First, arelapse model intended to vigorously investigate whether the antitumoractivity of CART19 cells was impacted by GM-CSF neutralization was used.NOD/SCID/interleukin-2 receptor gamma null (NSG) mice were injected with1×10⁶ luciferase⁺ NALM6 cells and then imaged 6 days later, allowingsufficient time for mice to achieve very high tumor burdens. Mice wererandomized to receive a single injection of either CART19 or UTD cellsand 10 days of either isotype control antibody or lenzilumab (FIG. 28A).GM-CSF assay on serum collected 8 days after CART19 injection revealedthat lenzilumab successfully neutralizes GM-CSF in the context of CART19therapy (FIG. 28B). Bioluminescence imaging one week after CART19injection showed that CART19 in combination with lenzilumab effectivelycontrolled leukemia in this high tumor burden relapse model andsignificantly better than control UTD cells (FIGS. 28C-28D). Treatmentwith CART19 in combination with lenzilumab resulted in potent anti-tumoractivity and improved overall survival, similar to CART19 with controlantibody despite neutralization of GM-CSF levels, indicating that GM-CSFdoes not impair CAR-T cell activity in vivo (FIG. 36). Second, theseexperiments were performed in a primary ALL patient derived xenograftmodel, in the presence of human PBMCs as this represents a more relevantheterogeneous model. After conditioning chemotherapy with busulfan, micewere injected with blasts derived from patients with relapsed ALL. Micewere monitored for engraftment for several weeks through serial tailvein bleedings and when the CD19⁺ blasts in the blood were approximately1/uL, mice were randomized to receive CART19 treatment in combinationwith PBMCs with either lenzilumab plus an anti-mouse GM-CSFneutralization antibody or isotype control IgG antibodies starting onthe day of CART 19 injection for 10 days (FIG. 28E). In this primary ALLxenograft model, GM-CSF neutralization in combination with CART19therapy resulted in a significant improvement in leukemic diseasecontrol sustained over time for at least 35 days post CART19administration as compared to CART19 plus isotype control (FIG. 28F).This suggests that GM-CSF neutralization may play a role in reducingrelapses and increasing durable complete responses after CART19 celltherapy.

Example 20

Generation of GM-CSF^(k/o) CART19

A guide RNA (gRNA) targeting exon 3 of human GM-CSF was selected viascreening gRNAs previously reported to have high efficiency for humanGM-CSF, as described in Sanjana N E et al., Improved vectors andgenome-wide libraries for CRISPR screening. Nature Methods. 2014;11(8):783-784. Prepublished on 2014 Jul. 31 as DOI 10.1038/nmeth.3047,which is hereby incorporated by reference in its entirety. This gRNA wasordered in a CAS9 third generation lentivirus construct (lentiCRISPRv2),controlled under a U6 promotor (GenScript, Township, N.J., USA).Lentiviral particles encoding this construct were produced as describedabove. T cells were dual transduced with CAR19 andGM-CSFgRNA-lentiCRISPRv2 lentiviruses, 24 hours after stimulation withCD3/CD28 beads. CAR-T cell expansion was then continued as describedabove. To analyze efficiency of targeting GM-CSF, genomic DNA wasextracted from the GM-CSF^(k/o) CART19 cells using PureLink Genomic DNAMini Kit (Invitrogen, Carlsbad, Calif., USA). The DNA of interest wasPCR amplified using Choice Taq Blue Mastermix (Thomas Scientific,Minneapolis, Minn., USA) and gel extracted using QIAquick Gel ExtractionKit (Qiagen, Germantown, Md., USA) to determine editing. PCR ampliconswere sent for Eurofins sequencing (Louisville, Ky., USA) and allelemodification frequency was calculated using TIDE (Tracking of Indels byDecomposition) a method that requires only two parallel PCR reactionsfollowed by a pair of standard capillary sequencing analyses; the tworesulting sequencing traces are then analyzed using specially designedsoftware that is provided as a simple web tool and as R code availableat tide.nki.nl, as described by Brinkman E K, et al., Easy quantitativeassessment of genome editing by sequence trace decomposition. NucleicAcids Research. 2014; 42(22):e168. Prepublished on 2014 Oct. 11 as DOI10.1093/nar/gku936, which is incorporated herein by reference in itsentirety. FIG. 34B describes the gRNA sequence and primer sequences, andFIG. 34A(i)-34A(iii) depicts the schema for generation of GM-CSF^(k/o)CART19 schema.

Example 21

GM-CSF CRISPR Knockout CAR-T Cells Exhibit Reduced Expression of GM-CSF,Similar Levels of Key Cytokines and Chemokines, and Enhanced Anti-TumorActivity

To confidently exclude any role for GM-CSF critical in CAR-T cellfunction, the GM-CSF gene was disrupted during CAR-T cell manufacturingusing a gRNA that has been reported to yield high efficiency knockoutand is cloned into a CRISPR lentivirus backbone, as described by SanjanaN E, et al., Nature Methods. 2014; 11(8):783-784. Prepublished on 2014Jul. 31 as DOI 10.1038/nmeth.3047, which is hereby incorporated byreference in its entirety. Using this gRNA, we achieved around 60%knockout efficiency in CART19 cells (FIG. 37). When CAR-T cells werestimulated with the CD19⁺ cell line NALM6, GM-CSF^(k/o) CAR-T cellsproduced statistically significantly less GM-CSF compared to CART19 witha wild-type GM-CSF locus (“wild type CART19 cells”). GM-CSF knockout inCAR-T cells did not impair the production of other key T cell cytokines,including IFN-γ, IL-2, or CAR-T cell antigen specific degranulation(CD107a) (FIG. 29A) but did exhibit reduced expression of GM-CSF (FIG.29B). To confirm that GM-CSF^(k/o) CAR-T cells continue to exhibitnormal functions, their in vivo efficacy in the high tumor burdenrelapsing xenograft model of ALL was tested (as described in FIG. 28A).In this xenograft model, utilization of GM-CSF^(k/o) CART19 instead ofwild type CART19 markedly reduced serum levels of human GM-CSF at 7 daysafter CART19 treatment (FIG. 29B). Bioluminescence imaging data impliedthat GM-CSF^(k/o) CART19 cells show enhanced leukemic control comparedto CART19 in this model (FIG. 38). Importantly, GM-CSF^(k/o) CART19cells demonstrated significant improvement in overall survival comparedto wild type CART19 cells (FIG. 29C). Human GM-CSF was statisticallysignificantly decreased via t test in the GM-CSF^(k/o) CART19 cellscompared to wild type CART19 (FIG. 29D). The mouse GM-CSF visuallyappears increased although this is not statistically significant via ttest (P=0.472367) (FIG. 29E). This lack of mouse GM-CSF reduction is notnecessarily surprising as the GM-CSF^(k/o) CART19 cells (which arehuman) are the only cells within the mouse that possess the knockout,thus mouse GMCSF would not likely be affected directly. By visualinspection, mouse IP-10, a chemokine that attracts numerous cell typesincluding T cells and monocytes, appears paradoxically increased inGM-CSF^(k/o) CART19 compared to CART19, but this is also notstatistically significant, P=0.4877 by t test (FIG. 29E). By visualinspection, mouse MIP1α (an inflammatory cytokine important inneutrophil attraction) and mouse M-CSF (a cytokine critical inmacrophage differentiation) appear reduced, although they are notstatistically significant with P=0.2437 and P=0.3619 (FIG. 29E). MouseIL-1b, a critical inflammatory cytokine produced by macrophages, andmouse IL-15, a cytokine produced by macrophages that aids in NK cellproliferation, appear reduced in GMCSF^(k/o) CART19 compared to CART19(FIG. 29E) with P values of P=0.0741 and P=0.0900, respectively (FIG.29E). Critical human T cell cytokines were not inhibited by GM-CSF^(k/o)(FIG. 29D). It should be emphasized that these xenografts were producedwith high burdens of the NALM6 cell line, and our CRS/NI model (FIGS.30A-30D, 31, 32A-32D and 33A-33D) require the use of primary ALL cellsto be generated. Thus, cytokine profiles unsurprisingly differ betweenthe two models as the NALM6 xenografts (FIGS. 29A-29E) do not developCRS or NI. Together, in the context of a NALM6 high tumor burden modelwithout CRS, results of FIGS. 29A-29E confirm FIGS. 27A-27D and 28A-28F,indicating that GM-CSF depletion does not impair normal cytokines orchemokines that are critical to CAR-T efficacy functions. In addition,the results in FIGS. 29A-29E indicate that GM-CSF^(k/o) CART mayrepresent a therapeutic option for “built in” GM-CSF control as amodification during CAR-T cell manufacturing.

Example 22

Patient Derived Xenograft Model for Neuro-Inflammation (NI) and CytokineRelease Syndrome/GM-CSF Neutralization In Vivo Ameliorates CytokineRelease Syndrome and neuroinflammation after CART19 therapy in axenograft model

Primary Patient-Derived ALL Xenografts

To establish primary ALL xenografts, NSG mice first received 30 mg/kgbusulfan IP (Selleckchem, Houston, Tex., USA). The following day, micewere injected with 2.5×10⁶ primary blasts derived from the peripheralblood of patients with relapsed or refractory ALL. Mice were monitoredfor engraftment for ˜10-13 weeks. When CD19⁺ cells were consistentlyobserved in the blood (approximately 1 cell/uL), they were randomized toreceive different treatments of CART19 (2.5×10⁶ cells IV) and PBMCsderived from the same donor (1×10⁵ cells IV) with or without antibodytherapy (10 mg/kg lenzilumab or isotype control IP for a total of 10days, starting on the day they received CAR-T cell therapy). Mice wereperiodically monitored for leukemic burden via tail vein bleeding.

Primary Patient-Derived ALL Xenografts for CRS/NI

Similar to the experiments above, mice were IP injected with 30 mg/kgbusulfan (Selleckchem, Houston, Tex., USA). The following day, micereceived 1-3×10⁶ primary blasts derived from the peripheral blood ofpatients with relapsed ALL. Mice were monitored for engraftment for˜10-13 weeks via tail vein bleeding. When serum CD19⁺ cells were ≥10cells/ul, the mice received CART19 (2-5×10⁶ cells IV) and commencedantibody therapy for a total of 10 days, as indicated. Mice were weighedon a daily basis as a measure of their well-being. Mouse brain MRIs wereperformed 5-6 days post CART19 injection and tail vein bleeding forcytokine/chemokine and T cell analysis was performed 4-11 days postCART19 injection.

MRI Acquisition

A Bruker Avance II 7 Tesla vertical bore small animal MRI system (BrukerBiospin) was used for image acquisition to evaluate central nervoussystem (CNS) vascular permeability. Inhalation anesthesia was inducedand maintained via 3 to 4% isoflurane. Respiratory rate was monitoredduring the acquisition sessions using an MRI compatible vital signmonitoring system (Model 1030; SA Instruments, Stony Brook, N.Y.). Micewere given an IP injection of gadolinium using weight-based dosing of100 mg/kg, and after a standard delay of 15 min, a volume acquisitionT1-weighted spin echo sequence was used (repetition time=150 ms, echotime=8 ms, field of view: 32 mm×19.2 mm×19.2 mm, matrix: 160×96×96;number of averages=1) to obtain T1-weighted images. Gadolinium-enhancedMRI changes were indicative of blood-brain-barrier disruption.²⁴Volumetric analysis was performed using Analyze Software packagedeveloped by the Biomedical Imaging Resource at Mayo Clinic.

Results

In this model, conditioned NSG mice were engrafted with primary ALLblasts and monitored for engraftment for several weeks until theydeveloped high disease burden (FIG. 30A). When the level of CD19⁺ blastsin the peripheral blood was >10/uL, mice were randomized to receivedifferent treatments as indicated (FIG. 30A). Treatment with CART19(with control IgG antibodies or with GM-CSF neutralizing antibodies)successfully eradicated the disease (FIG. 30B). Within 4-6 days aftertreatment with CART19, mice began to develop motor weakness, hunchedbodies, and progressive weight loss; symptoms consistent with CRS andNI. This was associated with elevation of key serum cytokines andchemokines 4-11 days post CART19 injection similar to what is seen inhuman CRS after CAR-T cell therapy (including human GM-CSF, TNF-α,IFN-γ, IL-10, IL-12, IL-13, IL-2, IL-3, IP-10, MDC, MCP-1, MIP-1α,MIP-1β, and mouse IL-6, GM-CSF, IL-4, IL-9, IP-10, MCP-1, and MIG).These mice treated with CART19 also developed NI as indicated by brainMRI analyses revealing abnormal T1 enhancement, suggestive ofblood-brain barrier disruption and possibly brain edema (FIG. 30C),together with flow cytometric analysis of harvested brains revealinginfiltration of human CART19 cells (FIG. 30D). In addition, RNA-seqanalyses of brain sections harvested from mice that developed thesesigns of NI showed significant upregulation of genes regulating the Tcell receptor, cytokine receptors, T cell immune activation, T celltrafficking, and T cell and myeloid cell differentiation (FIG. 31, Table6).

TABLE 6 Table of canonical pathways altered in brains from patientderived xenografts after treatment with CART19 cells in tabular format.Conical Pathway Adj P-Value Genes regulation of 9.45E−14 IFITM1, ITGB2,TRAC, ICAM3, CD3G, immune response PTPN22, CD3E, ITGAL, SAMHD1, SLA2,(GO: 0050776) CD3D, ITGB7, SLAMF6, B2M, NPDC1, CD96, BTN3A1, ITGA4,SH2D1A, HLA- B, HLA-C, BTN3A2, HLA-A, CD8B, SELL, CD8A, CD226, CD247,CLEC2D, HCST, BIRC3 cytokine-mediated 1.36E−12 IFITM1, SP100, TRADD,ITGB2, IL2RG, signaling SAMHD1, IL27RA, OASL, CNN2, pathway IL18RAP,RIPK1, CCR5, IL12RB1, B2M, (GO: 0019221) GBP1, IL6R, JAK3, CCR2, IL32,ANXA1, IL4R, TGFB1, IL10RB, IL10RA, STAT2, PRKCD, HLA-B, HLA-C, IL16,HLA-A, TNFRSF1B, CD4, IRF3, OAS2, IL2RB, FAS, TNFRSF25, LCP1, P4HB,IL7R, MAP3K14, CD44, IL18R1, IRF9, MYD88, BIRC3 T cell receptor 1.30E−11ZAP70, CD4, CD6, CD8B, CD8A, CD3G, complex CD247, CD3E, CD3D, CARD11(GO: 0042101) T cell activation 2.07E−11 ITK, RHOH, CD3G, NLRC3, PTPN22,(GO: 0042110) CD3E, SLA2, CD3D, CD2, ZAP70, CD4, PTPRC, CD8B, CD8A, LCK,CD28, LCP1, LAT regulation of T cell 2.46E−10 PTPN22, LAX1, CCDC88B,CD2, CD4, activation LCK, SIT1, TBX21, TIGIT, JAK3, LAT, (GO: 0050863)PAG1, CCR2 T cell receptor 4.35E−08 ITK, BTN3A1, TRAC, WAS, CD3G,signaling pathway PTPN22, BTN3A2, CD3E, CD3D, ZAP70, (GO: 0050852) CD4,PTPRC, LCK, GRAP2, LCP2, CD247, CARD11, LAT, PAG1 positive regulation1.57502E−07   GBP5, ANXA1, TGFB1, CYBA, PTPN22, of cytokine PARK7,TMEM173, CCDC88B, MAVS, production CD6, IRF3, CD28, RIPK1, SLAMF6, (GO:0001819) CD46, IL12RB1, TIGIT, IL6R, CARD11, MYD88, CCR2 T cell 2.36E−07ZAP70, CD4, ANXA1, PTPRC, CD8A, differentiation LCK, CD28, RHOH, PTPN22,CD3D (GO: 0030217) cytokine receptor 2.43E−07 IL4R, IL10RB, IL10RA,IL2RG, CD4, activity CXCR3, IL2RB, CCR5, IL12RB1, IL7R, (GO: 0004896)IL6R, CD44, CCR2 type I interferon 3.27E−07 IFITM1, SP100, IRF3, OAS2,STAT2, signaling HLA-B, HLA-C, HLA-A, SAMHD1, IRF9, pathway MYD88, OASL(GO: 0060337) response to cytokine 0.0004679 SIGIRR, IFITM1, SP100,HCLS1, RIPK1, (GO: 0034097) PTPN7, IKBKE, IL6R, JAK3, IL18R1, MYD88, AESregulation of innate 0.001452 GBP5, GFI1, STAT2, ADAM8, NLRC3, immunePTPN22, SAMHD1, BIRC3 response (GO: 0045088) regulation of tumor0.003843 CD2, MAVS, CYBA, NLRC3, PTPN22, necrosis RIPK1, SLAMF1 factorproduction (GO: 0032680) T cell receptor 0.0102397 LCK, CD3G, CD3Ebinding (GO: 0042608) regulation of tumor 0.0124059 SHARPIN, TRADD,CASP4, RIPK1, necrosis TRAF1, BIRC3 factor-mediated signaling pathway(GO: 0010803) Positive regulation 0.0376647 CD4, HCLS1, RIPK1, EVI2B ofmyeloid Leukocyte differentiation (GO: 0002763)

Using the xenograft patient derived model for NI and CRS shown in FIG.30A, the effect of GM-CSF neutralization on CART19 toxicities wasinvestigated. To rule out the cofounding effect of mouse GM-CSF, micereceived CART19 cells in combination with 10 days of GM-CSF antibodytherapy (10 mg/kg lenzilumab and 10 mg/kg antimouse GM-CSF neutralizingantibody) or isotype control antibodies. GM-CSF neutralizing antibodytherapy statistically significantly reduced CRS induced weight lossafter CART19 therapy (FIG. 32A). Cytokine and chemokine analysis 11 daysafter CART19 cell therapy showed that human GM-CSF was neutralized bythe antibody (FIG. 32B). In addition, GM-CSF neutralization resulted insignificant reduction of several human (IP-10, IL-3, IL-2, IL-1Ra,IL-12p40, VEGF, GM-CSF) (FIG. 32C) and mouse (MIG, MCP-1, KC, IP-10)(FIG. 32D) cytokines and chemokines. Interferon gamma-induced protein(IP-10, CXCL10) is produced by monocytes among other cell types andserves as a chemoattractant for numerous cell types including monocytes,macrophages, and T cells. IL-3 plays a role in myeloid progenitordifferentiation. IL-2 is a key T cell cytokine. Interleukin-1 receptorantagonist (IL-1Ra) inhibits IL-1. (IL-1 is produced by macrophages andis a family of critical inflammatory cytokines.) IL-12p40 is a subunitof IL-12, which is produced by macrophages among other cell types andcan encourage Th1 differentiation. Vascular endothelial growth factor(VEGF) encourages blood vessel formation. Monokine induced by gammainterferon (MIG, CXCL9) is a T cell chemoattractant. Monocytechemoattractant protein 1 (MCP-1, CCL2) attracts monocytes, T cells, anddendritic cells. KC (CXCL1) is produced by macrophages among other celltypes and attracts myeloid cells such as neutrophils. There was also anon-statistically significant reduction of several other human and mouecytokines and chemokines after GM-CSF neutralization. This suggests thatGMCSF plays a role in the downstream activity of several cytokines andchemokines that are instrumental in the cascade that results in CRS andNI.

Brain MRIs 5 days after CAR19 treatment showed that GM-CSFneutralization reduced T1 enhancement as a measure of braininflammation, blood-brain barrier disruption, and possibly edema,compared to CART19 plus control antibodies. The MRI images after GM-CSFneutralization (with lenzilumab and anti-mouse GM-CSF antibody) weresimilar to baseline pre-treatment scans, suggesting that GM-CSFneutralization effectively helped abrogate the NI associated with CART19therapy (FIGS. 33A and 33B). Using human ALL blasts and human CART19 inthis patient-derived xenograft model, GM-CSF neutralization after CART19reduced neuro-inflammation by 75% compared to CART19 plus isotypecontrols (FIG. 33B). This is a significant finding and the first time ithas been demonstrated in vivo that the NI caused by CART19 can beeffectively abrogated. Human CD3 T cells were present in the brain afterCART19 therapy as assayed by flow cytometry, and with GM-CSFneutralization, there was a difference in raw average with reduction inbrain CD3 T cells, but it did not meet statistical significance (FIGS.33C, 30D, and 39). Finally, a difference in raw average (although thisdid not reach statistical significance) with reduction of CD11b+ brightmacrophages was observed in the brains of mice receiving GM-CSFneutralization during CAR-T cell therapy compared to isotype controlduring CAR-T therapy (FIG. 33D), possibly implicating that GM-CSFneutralization helps reduce macrophages within the brain.

The results of Examples 18-19 and 22 demonstrate that neutralization ofGM-CSF abrogates toxicities after CAR-T cell therapy and may enhancetheir therapeutic activity. Specifically, it was shown that GM-CSFneutralization in combination with CART19 therapy prevents thedevelopment of CRS and significantly reduces the severity of NI in axenograft model using human ALL blasts and human CART19. GM-CSFneutralization resulted in a reduction in chemokines associated withmyeloid trafficking, such as IP-10, MCP-1, KC, and other inflammatorycytokines and chemokines, and is associated with decreased raw averages(although not statistically significant) of T cell infiltration andmyeloid cell activation in the brain. Intriguingly, the experimentsherein also suggest that GM-CSF inhibition enhances CART19proliferation, anti-tumor activity, and overall survival in vivo. Basedon these results, GM-CSF neutralization can be viewed as a potentialnext generation strategy to enable routine CAR-T cellular immunotherapy.

In the studies described herein, GM-CSF neutralization with lenzilumabdid not impair any CART19 effector functions in vitro. In two differentxenograft models (NALM6 xenografts and patient derived xenografts),CART19 combined with lenzilumab effectively eradicated the tumor despiteGM-CSF neutralization and significantly improved leukemic diseasecontrol 35 days post-treatment while CART19 plus isotype control couldnot maintain disease control after 35 days. Lastly, in Examples 21-22,GM-CSF^(k/o) CART19 cells exhibited potent effector functions in vitroand demonstrated significantly improved overall survival compared toCART19 in vivo.

The herein described CRS and NI model is a unique and relevant ALLpatient derived xenograft model for the development of therapies fortoxicities after human CAR-T cell therapy. In the model described here,the time interval between CAR-T cell infusion to onset of symptoms,brain MRI changes, cytokine and chemokine elevation, and infiltration ofeffector cells into the CNS are all similar to what is reported inpatients that develop toxicities after CART19 therapy. Mice developedsymptoms of CRS and NI (weight loss, decline in motor function, andhunched bodies). Changes in brain MRI were detected 4-6 days afterinfusion of CART19 cells. Brain MRI T1 uptake is suggestive ofblood-brain barrier disruption and possibly brain edema and iscomparable to changes noted on human brain MRI in cases of severeneurotoxicity, as described by Gust et al. 2017 Cancer Discovery. 2017;7(12):1404-1419. Prepublished on 2017 Oct. 14 as DOI10.1158/2159-8290.CD-17-0698, which is incorporated herein by referencein its entirety.

Interestingly, Gust et al. 2017 further describes that blood brainbarrier permeability prevented protection of the CSF from systemiccytokines, which induced vascular pericyte stress and secretion ofendothelium-activating cytokines, and patients showed evidence ofendothelial activation. In the CRS/NI model described herein, GM-CSF wasfound to be neutralized in the serum of mice receiving CART19 therapywith GM-CSF neutralizing antibodies compared to CART19 and isotypecontrol antibodies. Thus, T cells within the mouse brains themselvescould provide GM-CSF production, and serum GM-CSF among other cytokinesand chemokines were possibly able to reach the CSF. In addition,endothelium cells are able to produce GM-CSF, which may result in acycle of exacerbation. NI was associated with infiltration of T cellsand activation of myeloid cells in the CNS, similar to CSF changes inpatients with CAR-T induced neurotoxicity, as well as in non-humanprimate models. The herein described model is similar to previouslyreported patient derived xenograft models where CRS developed afterCAR-T cell therapy. A recent report suggested that blockade of IL-1prevents NI through the depletion of myeloid cells. However, thedevelopment of NI in that model was delayed and related to meningealthickening, unlike what was observed in the model described herein andin patients receiving CART19 therapy. Therefore, the model describedherein is provided as a reliable way to investigate novel interventionsfor the prevention and treatment of CRS and neurotoxicity after CART19cell therapy. The results described herein show that GM-CSFneutralization results in a reduction in key myeloid and severalinflammatory cytokines and chemokines, suggesting that GM-CSF is acritical cytokine in downstream activation of several cytokines andchemokines; blockade contributes to a decrease in raw averages inmyeloid and T cell infiltration in the brain/CNS (although statisticalsignificance was not reached); and blockade helps reduceneuro-inflammation of apparent neurotoxicities.

Interestingly, an exponential increase in CART19 cell proliferation wasobserved, enhanced anti-tumor activity, and improved overall survivalwith GM-CSF blockade. For example, CART19 antigen specific proliferationin the presence of monocytes increased in vitro after GM-CSFneutralization. Moreover, in ALL patient derived xenografts, CART19cells resulted in a more durable disease control when combined withlenzilumab. In addition, it was found that GM-CSF^(k/o) CAR-T cells weremore effective in controlling leukemia in NALM6 xenografts anddemonstrated improved overall survival. While the mechanisms forenhanced CART effector functions after GM-CSF depletion are currentlyunclear, the results provided herein are consistent with previousreports indicating that monocytes impair T cell expansion ex vivo andthat M2 polarized macrophages inhibit CART19 antigen specificproliferation. This is an important finding because across CAR-Tclinical trials, improved CAR-T cell proliferation was consistentlyassociated with improved efficacy and response (i.e., overall andcomplete response rates).

It is known that activated T cells produce GM-CSF. T cells do notpossess all the subunits for the GM-CSF receptor, so in ordinarycircumstances, GM-CSF does not normally feedback on T-cells directly,although it can under some circumstances at very high levels. Instead,this GM-CSF affects the behaviors of numerous other cell types includingmacrophages and dendritic cells. The subsequent activation of thesecells results in actions that work to stimulate T cells such as cytokineproduction and antigen presentation. T cell stimulation can furtherdrive production of GM-CSF and other cytokines to in turn act on theother cell types like macrophages and dendritic cells, which drives thecycle. In CAR-T cell therapy, it is likely that the large number ofactivated T cells produced over a very short timeline pushes this cycleto an extreme situation. The results described herein suggest thatblocking GM-CSF helps prevent this immune overstimulation withoutimpairing T cell functions, actually enhancing them. The exactmechanisms for enhanced CAR-T cell effector functions after GM-CSFblockade are unclear.

Finally, the results provided herein results additionally suggest thatthe development of GM-CSF^(k/o) CART19 cells may represent a novel wayto partially control GM-CSF production that can be incorporated intocurrent CAR-T cell manufacturing. These results indicate that thesecells function normally and could represent an independent therapeuticapproach to enhance the therapeutic window after CAR-T cell therapy. Ananti-GM-CSF antibody, such as lenzilumab, is a clinical stagetherapeutic solution to neutralize GM-CSF, abrogate both CRS andneuro-inflammation of apparent neurotoxicities, and potentially improveCAR-T cell function.

The studies described herein represent a significant advance inunderstanding and preventing toxicities after CAR-T cell therapy. Theseresults strongly suggest that modulating myeloid cell behavior throughGM-CSF blockade helps control CAR-T cell mediated toxicities and reducetheir immunosuppressive features to improve leukemic control. Thesestudies illuminate a novel approach to abrogate neuro-inflammation ofapparent neurotoxicities and CRS through GM-CSF neutralization that alsopotentially enhances CAR-T cell functions.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

All publications, accession numbers, patents, and patent applicationscited in this specification are herein incorporated by reference as ifeach was specifically and individually indicated to be incorporated byreference.

Exemplary V_(H) Region Sequences of Anti-GM-CSF Antibodies of theInvention:

(VH#1, FIG. 1) SEQ ID NO: 1QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCVRRD RFPYYFDYWGQGTLVTVSS(VH#2, FIG. 1) SEQ ID NO: 2QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYIHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVAITRDTSASTAYMELSSLRSEDTAVYYCARRD RFPYYFDYWGQGTLVTVSS(VH#3, FIG. 1) SEQ ID NO: 3QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYIHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVAITRDTSASTAYMELSSLRSEDTAVYYCARRQ RFPYYFDYWGQGTLVTVSS(VH#4, FIG. 1) SEQ ID NO: 4QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYIHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVAITRDTSASTAYMELSSLRSEDTAVYYCVRRQ RFPYYFDYWGQGTLVTVSS(VH#5, FIG. 1) SEQ ID NO: 5QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYIHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCVRRQ RFPYYFDYWGQGTLVTVSSExemplary V_(L) Region Sequences of Anti-GM-CSF Antibodies of theInvention:

(VK#1, FIG. 1) SEQ ID NO: 6EIVLTQSPATLSVSPGERATLSCRASQSVGTNVAWYQQKPGQAPRVLIYSTSSRATGITDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQFNRSPLTFGG GTKVEIK(VK#2, FIG. 1) SEQ ID NO: 7EIVLTQSPATLSVSPGERATLSCRASQSVGTNVAWYQQKPGQAPRVLIYSTSSRATGITDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQFNKSPLTFGG GTKVEIK(VK#3, FIG. 1) SEQ ID NO: 8EIVLTQSPATLSVSPGERATLSCRASQSIGSNLAWYQQKPGQAPRVLIYSTSSRATGITDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQFNRSPLTFGG GTKVEIK(VK#4, FIG. 1) SEQ ID NO: 9EIVLTQSPATLSVSPGERATLSCRASQSIGSNLAWYQQKPGQAPRVLIYSTSSRATGITDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQFNKSPLTFGG GTKVEIKExemplary kappa constant region SEQ ID NO: 10RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGECExemplary heavy chain constant region, f-allotype: SEQ ID NO: 11ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

What is claimed is:
 1. A method for reducing relapse rate or preventingoccurrence of tumor relapse in a subject treated with immunotherapy, themethod comprising administering to the subject a recombinant hGM-CSFantagonist, wherein the recombinant hGM-CSF antagonist is anti-hGM-CSFantibody lenzilumab.
 2. The method of claim 1, wherein saidimmunotherapy comprises adoptive cell transfer, administration ofmonoclonal antibodies, administration of cytokines, administration of acancer vaccine, T cell engaging therapies, or any combination thereof.3. The method of claim 2, wherein the adoptive cell transfer comprisesadministering chimeric antigen receptor-expressing T-cells (CART-cells), T-cell receptor (TCR) modified T-cells, tumor-infiltratinglymphocytes (TIL), chimeric antigen receptor (CAR)-modified naturalkiller cells, or dendritic cells, or any combination thereof.
 4. Themethod of claim 1, wherein the anti-hGM-CSF antibody lenzilumab binds ahuman GM-CSF.
 5. The method of claim 1, wherein the anti-hGM-CSFantibody lenzilumab binds a primate GM-CS F.
 6. The method of claim 1,wherein the anti-hGM-CSF antibody lenzilumab binds a mammalian GM-CSF.7. The method of claim 1, wherein the anti-hGM-CSF antibody lenzilumabis a monoclonal antibody.
 8. The method of claim 1, wherein theanti-hGM-CSF antibody lenzilumab is a human GM-CSF neutralizingantibody.
 9. The method of claim 1, wherein the anti-hGM-CSF antibodylenzilumab is a recombinant antibody.
 10. The method of claim 1, whereinthe anti-hGM-CSF antibody lenzilumab is an engineered human antibody.11. The method of claim 1, wherein the anti-hGM-CSF antibody lenzilumabbinds to the same epitope as chimeric 19/2.
 12. The method of claim 1,wherein the anti-hGM-CSF antibody lenzilumab comprises a VH region thatcomprises a CDR3 binding specificity determinant RQRFPY (SEQ ID NO: 12),a J segment, and a V-segment, wherein the J-segment comprises at least95% identity to human JH4 (YFDYWGQGTLVTVSS) (SEQ ID NO: 14) and theV-segment comprises at least 90% identity to a human germ line VHl 1-03sequence.
 13. The method of claim 12, wherein the J segment comprisesYFDYWGQGTLVTVSS (SEQ ID NO: 14).
 14. The method of claim 12, wherein theCDR3 comprises RQRFPYYFDY (SEQ ID NO: 15).
 15. The method of claim 12,wherein the anti-hGM-CSF antibody lenzilumab comprises a VH CDR1 and aVH CDR2 as shown in a VH region having a sequence of VH #5 set forth inFIG.
 1. 16. The method of claim 12, wherein the V-segment sequence has aVH V segment sequence of VH #5 shown in FIG.
 1. 17. The method of claim12, wherein the VH has the sequence of VH #5 set forth in FIG.
 1. 18.The method of claim 1, wherein the anti-hGM-CSF antibody lenzilumabcomprises a VL-region that comprises a CDR3 comprising the amino acidsequence FNK.
 19. The method of claim 18, wherein the anti-hGM-CSFantibody lenzilumab comprises a VL region that comprises a CDR3comprising QQFNKSPLT (SEQ ID NO: 18).
 20. The method of claim 11, wherethe VL region comprises both a CDR1 and CDR2 of a VL region having thesequence of VK #2 shown in FIG.
 1. 21. The method of claim 18, whereinthe VL region comprises a V segment that has at least 95% identity tothe VKIII A27 V-segment sequence as shown in FIG.
 1. 22. The method ofclaim 18, wherein the VL region has the sequence of VK #2 set forth inFIG.
 1. 23. The method of claim 1, wherein the anti-hGM-CSF antibodylenzilumab has a VH region CDR3 binding specificity determinant RQRFPY(SEQ ID NO: 12) and a VL region that has a CDR3 comprising QQFNKSPLT(SEQ ID NO: 18).
 24. The method of claim 1, wherein the anti-hGM-CSFantibody lenzilumab has a VH region having sequence VH #5 set forth inFIG. 1 and a VL region having sequence VK #2 set forth in FIG.
 1. 25.The method of claim 24, wherein the VH region or the VL region, or boththe VH and VL region amino acid sequences comprise a methionine at theN-terminus.
 26. The method of claim 3, wherein the CAR-T cells are CD19CAR-T cells.
 27. The method of claim 1, wherein the reducing relapserate or preventing occurrence of tumor relapse in the subject occurs inan absence of an incidence of immunotherapy-related toxicity.
 28. Themethod of claim 1, wherein the reducing relapse rate or preventingoccurrence of tumor relapse in the subject occurs in a presence of anincidence of immunotherapy-related toxicity.
 29. The method of claim 28,wherein the immunotherapy-related toxicity is CAR-T related toxicity.30. The method of claim 29, wherein the CAR-T related toxicity iscytokine release syndrome, neurotoxicity or neuro-inflammation.
 31. Themethod of claim 1, wherein the tumor relapse occurrence is reduced byfrom 50% to 100% in the first one-quarter of a year after administeringthe recombinant GM-CSF antagonist compared to tumor relapse occurrencein a subject treated with immunotherapy and not administered arecombinant GM-CSF antagonist.
 32. The method of claim 1, wherein thetumor relapse occurrence is reduced by from 50% to 95% in the firsthalf-year after administering the recombinant GM-CSF antagonist.
 33. Themethod of claim 1, wherein the tumor relapse occurrence is reduced byfrom 50% to 90% in the first year after administering the recombinantGM-CSF antagonist.
 34. The method of claim 1, wherein the tumor relapseoccurrence is prevented long-term.
 35. The method of claim 1, whereinthe tumor relapse occurrence is prevented by 12-36 months.
 36. Themethod of claim 1, wherein the tumor relapse occurrence is preventedcompletely (100%).
 37. The method of claim 1, wherein the subject hasacute lymphoblastic leukemia.